Rejuvenation or preservation of germ cells

ABSTRACT

Certain embodiments disclosed herein include, but are not limited to, at least one of compositions, methods, devices, systems, kits, or products regarding rejuvenation or preservation of germ cells or gametes. Certain embodiments disclosed herein include, but are not limited to, methods of modifying germ cells or gametes, or methods of administering modified germ cells or gametes to at least one biological tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and claims the benefit of theearliest available effective filing date(s) from the following listedapplication(s) (the “Related Applications”) (e.g., claims earliestavailable priority dates for other than provisional patent applicationsor claims benefits under 35 USC §119(e) for provisional patentapplications, for any and all parent, grandparent, great-grandparent,etc. applications of the Related Application(s)). All subject matter ofthe Related Applications and of any and all parent, grandparent,great-grandparent, etc. applications of the Related Applications isincorporated herein by reference to the extent such subject matter isnot inconsistent herewith.

Related Applications:

-   -   For purposes of the USPTO extra-statutory requirements, the        present application constitutes a continuation-in-part of U.S.        patent application Ser. No. to be assigned, Docket No.        1004-002-013A-000000, entitled REJUVENATION OR PRESERVATION OF        GERM CELLS, naming Roderick A. Hyde, Edward K. Y. Jung and        Lowell L. Wood, Jr. as inventors, filed 24 Jun. 2010, which is        currently co-pending, or is an application of which a currently        co-pending application is entitled to the benefit of the filing        date.    -   For purposes of the USPTO extra-statutory requirements, the        present application constitutes a continuation-in-part of U.S.        patent application Ser. No. to be assigned, Docket No.        1004-002-013B-000000, entitled REJUVENATION OR PRESERVATION OF        GERM CELLS, naming Roderick A. Hyde, Edward K. Y. Jung and        Lowell L. Wood, Jr. as inventors, filed 24 Jun. 2010, which is        currently co-pending, or is an application of which a currently        co-pending application is entitled to the benefit of the filing        date.    -   For purposes of the USPTO extra-statutory requirements, the        present application constitutes a continuation-in-part of U.S.        patent application Ser. No. to be assigned, Docket No.        1004-002-013C-000000, entitled REJUVENATION OR PRESERVATION OF        GERM CELLS, naming Roderick A. Hyde, Edward K. Y. Jung and        Lowell L. Wood, Jr. as inventors, filed 24 Jun. 2010, which is        currently co-pending, or is an application of which a currently        co-pending application is entitled to the benefit of the filing        date.

The United States Patent Office (USPTO) has published a notice to theeffect that the USPTO's computer programs require that patent applicantsreference both a serial number and indicate whether an application is acontinuation or continuation-in-part. Stephen G. Kunin, Benefit ofPrior-Filed Application, USPTO Official Gazette Mar. 18, 2003, availableat http://www.uspto.gov/web/offices/com/sol/og/2003/week11/patbene.htm.The present Applicant Entity (hereinafter “Applicant”) has providedabove a specific reference to the application(s)from which priority isbeing claimed as recited by statute. Applicant understands that thestatute is unambiguous in its specific reference language and does notrequire either a serial number or any characterization, such as“continuation” or “continuation-in-part,” for claiming priority to U.S.patent applications. Notwithstanding the foregoing, Applicantunderstands that the USPTO's computer programs have certain data entryrequirements, and hence Applicant is designating the present applicationas a continuation-in-part of its parent applications as set forth above,but expressly points out that such designations are not to be construedin any way as any type of commentary and/or admission as to whether ornot the present application contains any new matter in addition to thematter of its parent application(s).

All subject matter of the Related Applications and of any and allparent, grandparent, great-grandparent, etc. applications of the RelatedApplications is incorporated herein by reference to the extent suchsubject matter is not inconsistent herewith.

SUMMARY

Disclosed herein include embodiments relating to compositions, methods,delivery devices, computer systems, program products, andcomputer-implemented methods related to modified reproductive cells. Inan embodiment, a method of modifying a reproductive cell comprisesactively introducing at least one exogenous energy supplying factor intoat least one intracellular compartment of a reproductive cell. In anembodiment, a method of restoring mitochondrial function in areproductive cell comprises administering to a subject containing atleast one reproductive cell, an amount and for a time sufficient toincrease endogenous ATP levels of at least one of the subject'sreproductive cells, of at least one of an angiotensin receptor blockeror an angiotensin converting enzyme inhibitor. In an embodiment, theangiotensin receptor inhibitor includes angiotensin-II receptor blocker(ARB).

In an embodiment, a method of modifying a reproductive cell comprisesproviding at least one of creatine, phosphorcreatine, or creatine kinaseto a reproductive cell. In an embodiment, the reproductive cell islocated in a subject. In an embodiment, a method for up-regulatingmitochondrial function in a reproductive cell comprises activelyintroducing at least one exogenous energy supplying factor into at leastone intracellular compartment of a reproductive cell.

In an embodiment, a method of modifying an oocyte comprises introducingat least one exogenous energy supplying factor into at least oneintracellular compartment of an oocyte. In an embodiment, theintroducing at least one exogenous energy supplying factor includesallowing at least one exogenous energy supply factor to diffuse into theat least one intracellular compartment of the oocyte. In an embodiment,the introducing at least one exogenous energy supplying factor includesliposomal introduction by way of endocytosis.

In an embodiment, a method of modifying a female reproductive cellcomprises introducing at least one exogenous energy supplying factorinto at least one intracellular compartment of a female reproductivecell. In an embodiment, the female reproductive cell includes at leastone of an oogonium, primary oocyte, secondary oocyte, ootid, ovum, polarbody, follicular cell, or cumulus cell.

In an embodiment, a method of modifying a reproductive cell comprisesintroducing at least one exogenous constituent into at least oneintracellular compartment of a reproductive cell, the exogenousconstituent configured to increase at least one endogenous energysupplying factor.

In an embodiment, a method of selecting at least one reproductive cellcomprises detecting at least one endogenous energy supplying factor inat least one reproductive cell, and scoring the at least onereproductive cell based on the detection.

In an embodiment, a modified reproductive cell is produced by theprocess of actively introducing at least one exogenous energy supplyingfactor into at least one intracellular compartment of a reproductivecell.

In an embodiment, a kit comprises a detection material responsive to atleast one reproductive cell indicator, and means for administering atleast one exogenous energy supplying factor to at least one reproductivecell.

In an embodiment, a delivery device comprises a housing including atleast one reservoir containing at least one composition including atleast one exogenous energy supplying factor; the reservoir configured toreceive, retain, and dispense at least a portion of the composition toat least one reproductive cell or tissue.

In an embodiment, a system comprises at least one computing device; atleast one delivery device configured to receive, retain and dispense atleast a portion of a composition including at least one energy supplyingfactor to at least one reproductive cell; and a recordable mediumincluding one or more instructions that when executed on the computingdevice cause the computing device to regulate dispensing of at least aportion of the composition.

In an embodiment, a computer program product comprises a recordablemedium bearing one or more instructions for regulating dispensing of atleast one delivery device to at least one reproductive cell, wherein thedelivery device includes a composition including at least one exogenousenergy supplying factor, and generating at least one output.

In an embodiment, a computer-implemented method comprises regulatingdispensing a composition from at least one delivery device to at leastone reproductive cell, the composition including at least one energysupplying factor.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a partial view of a particular embodiment of adelivery device disclosed herein.

FIG. 2 illustrates a partial view of various embodiments of the deviceof FIG. 1.

FIG. 3 illustrates a partial view of various embodiments of the deviceof FIG. 1.

FIG. 4 illustrates a partial view of various embodiments of the deviceof FIG. 1.

FIG. 5 illustrates a partial view of various embodiments of the deviceof FIG. 1.

FIG. 6 illustrates a partial view of various embodiments of the deviceof FIG. 1.

FIG. 7 illustrates a partial view of a particular embodiment of a systemdisclosed herein.

FIG. 8 illustrates a partial view of various embodiments of the systemof FIG. 7.

FIG. 9 illustrates a partial view of various embodiments of the systemof FIG. 7.

FIG. 10 illustrates a partial view of various embodiments of the systemof FIG. 7:

FIG. 11 illustrates a partial view of a particular embodiment of acomputer program product disclosed herein.

FIG. 12 illustrates a partial view of a particular embodiment of acomputer-implemented method disclosed herein.

FIG. 13 illustrates a partial view of a particular embodiment of thecomputer-implemented method of FIG. 12.

FIG. 14 illustrates a partial view of a particular embodiment of thecomputer-implemented method of FIG. 12.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

In most animal species, gametogenesis occurs directly through meiosis inthe gonads, following migration of primordial germ cells during earlydevelopment. The cells, gametogonium, then pass through various stagesof development, from primary gametocytes, to secondary gametocytes, togametid, and fmally to gametes.

Gametogenesis in plants occurs by mitosis in gametophytes that grow fromhaploid spores after sporic meiosis. The male plant gamete is producedinside the pollen grain, while the female gamete is produced inside theembryo sac of the ovule.

Rejuvenation or preservation of vigorous reproductive cells (forexample, germ cells, any of the various stages of gametogonium, as wellas the corresponding reproductive supporting cells) increases the rateof fertilization, implantation, or birth of viable offspring. Forexample, mammalian oocyte and sperm vigor or quality is considered to bethe main limiting factor for in vitro fertilization (IVF) procedures.(See, e.g., Wang, et al., J. Zhejiang Univ. Sci. B, vol. 10(7), pp.483-492 (2009); and Rossato, et al., Human Rep. vol. 14, no. 3, pp.694-697 (1999), each of which is incorporated herein by reference).

Reproductive cell vigor decreases with increasing age of the subject, aswell as with increasing duration ex vivo storage (for example, whenharvested for in vitro fertilization procedures). It is reported thatpoor reproductive cell quality or vigor corresponds to an increase inage-related dysfunctions, such as a decrease in mitochondrial membranepotential, increase in mitochondrial DNA (mtDNA) damage, increase inchromosomal aneuploidy, increase in the incidence of apoptosis, andchanges in mitochondrial gene expression. Id. It is further reportedthat any of these dysfunctions can cause developmental retardation orarrest of preimplantation embryos in mammals. Id.

According to published reports, the oxidative phosphorylation withinmitochondria provides a major source of ATP needed for energy by maturegametes. See, for example, Zhang, et al. Cell Res. vol. 16, pp. 841-850(2006), and Wang, et al. J. Zhejiang Univ. Sci. B, Ibid.; each of whichis incorporated herein by reference. It has been reported that a deficitof mitochondria-derived ATP impairs mammalian oocyte spindles duringmitosis, thereby contributing to developmental problems in thedeveloping embryo. See, e.g., Zhang, et al. Ibid.

In relation to ATP levels, it has been reported that spermatozoamotility is directly correlated to ATP levels synthesized bymitochondrial respiration and stored prior to activation. See, forexample, Perchec, et al., J. Cell Sci., vol. 108, pp. 747-753 (1995),which is incorporated herein by reference. It has also been reportedthat ATP levels decline with time during storage of spermatozoan cells.See, for example, Rep. Biol. vol. 9, no. 1, pp. 39-49 (2009). Finally,exogenous lactate has been reported to serve as an energy supply formitochondria, as it supports ATP production by gametes, particularly inregard to spermatocytes, spermatids, and spermatogonia. See, forexample, Erkkila, et al. Mol. Hum. Reprod. Abstract Vol. 8, pp. 109-117,(2002); and Grootegoed, et al. Biochim. et Biophys. Acta, vol. 767, No.2, pp. 248-256 (1984), each of which is incorporated herein byreference.

It has been reported that the mitochondrial intermembrane space includescreatine kinase, an enzyme that produces phosphor-creatine from ATP andcreatine. See, e.g., Wallimann, et al., Abstract, Biochem. J. vol. 281,pp. 21-40 (1992), which is incorporated herein by reference. As has beenreported, creatine kinase is an enzyme utilized for energy metabolism bycatalyzing the reversible transfer of a phosphoryl group fromphosphocreatine to ADP, making the creatine pathway critical forincreasing or maintaining the vigor of reproductive cells. See, e.g.,Fritz-Wolf, et al. Abstract, Nature vol. 381, pp. 341-345 (1996), whichis incorporated herein by reference.

In plants, ATP-levels of germinating seeds are reported to becorrelative to vigor. For example, under conditions of artificial aging,seed deterioration was reflected in ATP-levels long before loss ofviability could be detected by conventional germination tests. See, forexample, Lunn and Madsen, Physiol. Plant., vol. 53, no. 2, pp. 164-169(2006), which is incorporated herein by reference.

It has been reported that oocyte communication with the surroundingcumulus cells is also an important indicator for oocyte development. Id.The cumulus cells are responsible for nutrition supply of oocytes in thefinal phase of oocyte maturation.

Morphological characters of cumulus cells can be used to value oocytequality and its maturation. Id. For example, compact and completecumulus and bright and homogenous ooplasm are considered indicators ofhigh-quality immature oocytes, while cumulus cells with no more thanthree layers or dark and heterogeneous ooplasm indicates low-qualityimmature oocytes. Id.

In general, it is reported that during meiotic progression, oocytematuration is linked with cumulus expansion or apoptosis and the numberof cumulus cells attached to matured oocytes decreases with age. Id. Itis reported that mitochondrial redistribution and their oxidativeactivity are essential to the degree of cumulus cell apoptosis duringoocyte maturation. Id Thus, mitochondria play a role in the reproductivesupporting cells, as well as the developing gametogonium itself.

The mitochondrial DNA content in oocytes with normal fertilization rateis reportedly higher than that in abnormal oocytes, and aging of thesubject from which the oocytes originate has been reported to exert anegative influence on the process of mitochondriogenesis. Id. Inmammalian oocytes, mitochondria reportedly regulate calcium andsynthesize ATP, both of which are necessary for good quality oocytes.Id. This contributes to the decline of the vigor of the oocytes withincreasing age in animal subjects.

Likewise, in spermatogenesis, male germ cell death is reported to bemodulated by ATP production, particularly by the mitochondria in thematuring gamete cells as well as the supporting Sertoli cells. See, forexample, Erkkila, et al., Am. J. Physiol. Endocrinol. Metab. vol. 290,pp. E1145-E1154 (2006), which is incorporated herein by reference.

Thus, in an embodiment disclosed herein, increasing intracellular levelsof an energy supplying factor is conducive to the preservation orrestoration of characteristics of reproductive cells that are typicallyfound in healthy, young-aged subjects. In some instances, suchcharacteristics have declined, for example, due to age, illness, geneticdisorder, mitochondrial defect, or other causes.

In an embodiment, at least one energy supplying factor (such as ATP,creatine, lactate, etc.) is provided to a reproductive cell, whichincreases the intracellular levels of the energy supplying factor (e.g.,ATP). In an embodiment, the at least one energy supplying factor isdelivered to the environment or microenvironment of the reproductivecell. In an embodiment, the at least one energy supplying factor isintracellularly delivered to the reproductive cell.

For example, methods of modifying at least one reproductive cell includeintroducing at least one exogenous energy supplying factor into at leastone intracellular compartment of a reproductive cell. In an embodiment,the introducing the at least one exogenous energy supplying factorincludes actively or passively introducing the at least one exogenousenergy supplying factor.

In an embodiment, the actively introducing at least one exogenous energysupplying factor into at least one intracellular compartment of areproductive cell includes injecting or electroporating the reproductivecell.

As indicated herein, the at least one exogenous energy supplying factorincludes, but is not limited to, at least one of exogenous creatine,exogenous phosphocreatine, exogenous creatine kinase, exogenouschlorophyll, exogenous lactate, exogenous glucose or other carbohydrate,exogenous calcium, exogenous ATP, exogenous ADP, exogenous AMP, or aprecursor thereof.

In an embodiment, the intracellular compartment of a reproductive cellincludes at least one of a nucleolus, nucleus, ribosome, vesicle, roughendoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmicreticulum, mitochondria, vacuole, cytoplasm, lysosome, or centriole. Inan embodiment, the reproductive cell is located in at least one of insitu, in vitro, in vivo, in utero, in planta, in silico, or ex vivo.

In an embodiment, a method of modifying at least one reproductive cellincludes detecting at least one endogenous energy supplying factor inthe reproductive cell prior to delivering at least one exogenous energysupplying factor thereto. In an embodiment, the at least one endogenousenergy supplying factor is the same factor as the exogenous energysupplying factor. In an embodiment, a method of modifying at least onereproductive cell includes measuring at least one endogenous energysupplying factor in the reproductive cell subsequent to injecting atleast one exogenous energy supplying factor therein.

In an embodiment, the at least one exogenous energy supplying factor isinjected in response to the presence or level of at least one endogenousenergy supplying factor in the reproductive cell. In an embodiment, amethod of modifying the at least one reproductive cell includesdetecting or measuring the level of the at least one exogenous energysupplying factor in the reproductive cell. In an embodiment, the atleast one exogenous energy supplying factor is provided in response tothe presence or level of at least one exogenous energy supplying factorpreviously delivered to the reproductive cell. In an embodiment, the atleast one exogenous energy supplying factor is provided in response tothe presence or level of at least one reproductive cell indicator. In anembodiment, the at least one reproductive cell indicator includes atleast one indicator of a property of the reproductive cell; a propertyof administering the at least one energy supplying factor to thereproductive cell; a property of the modified reproductive cell; aproperty of administering at least one additional round of an energysupplying factor to a modified reproductive cell; reproductive cell ortissue apoptosis; reproductive cell division; reproductive cellcytoskeletal rearrangement; reproductive cell mitochondrial quality,quantity, or arrangement; reproductive cell fertilization success orfailure; or reproductive cell or tissue secretion.

In an embodiment, a method of modifying a reproductive cell includesselecting at least one reproductive cell for modifying by injecting theat least one exogenous energy supplying factor. In an embodiment, the atleast one reproductive cell is selected for modifying based on one ormore reproductive cell parameters. In an embodiment, the one or morereproductive cell parameters include at least one of reproductive cellsize, reproductive cell stage, reproductive cell quality, health of thesubject from which the reproductive cell originates, species of thesubject from which the reproductive cell originates, or storageconditions of the reproductive cell. In an embodiment, the storageconditions of the reproductive cell include at least one of duration ofstorage time, storage temperature, storage size, reproductive celldilution, or storage solution(s). In an embodiment, the reproductivecell includes at least one of a germ cell, gametogonium, or gamete. Inan embodiment, the reproductive cell includes at least one of anoogonium, primary oocyte, secondary oocyte, ootid, ovum, polar body,follicular cell, cumulus cell, spermatogonia, primary spermatocyte,secondary spermatocyte, spermatid, spermatozoa, Sertoli cell, or Leydigcell.

In an embodiment, a method of modifying at least one reproductive cellincludes selecting at least one reproductive cell for manipulation. Inan embodiment, manipulation includes utilizing the selected reproductivecell for at least one fertilization event. In an embodiment,manipulation includes at least one of cell membrane stripping, geneticmodification, freezing, fusing with another reproductive cell, or fusingwith a somatic cell. In an embodiment, selecting the at least onereproductive cell for manipulation is based at least in part on at leastone of detected exogenous energy supplying factor, or detectedendogenous energy supplying factor. In an embodiment, selecting the atleast one reproductive cell for manipulation is based at least in parton at least one of the size of the reproductive cell, cleavage rate ofthe reproductive cell, metabolic profile of the reproductive cell,genomic profile of the reproductive cell, transcriptomic profile of thereproductive cell, or proteomic profile of the reproductive cell.

In an embodiment, a method of restoring mitochondrial function in areproductive cell includes administering to a subject containing atleast one reproductive cell, an amount and for a time sufficient toincrease endogenous ATP levels of at least one of the subject'sreproductive cells, of at least one of an angiotensin receptor blockeror an angiotensin converting enzyme inhibitor. In an embodiment, theangiotensin receptor inhibitor includes angiotensin-II receptor blocker(ARB).

In an embodiment, a method of modifying a reproductive cell includesproviding at least one of creatine, phosphorcreatine, or creatine kinaseto a reproductive cell. In an embodiment, the reproductive cell islocated in a subject.

In an embodiment, a method for up-regulating mitochondrial function in areproductive cell includes actively introducing at least one exogenousenergy supplying factor into at least one intracellular compartment of areproductive cell.

In an embodiment, a method of modifying an oocyte includes introducingat least one exogenous energy supplying factor into at least oneintracellular compartment of an oocyte. In an embodiment, introducingincludes allowing at least one exogenous energy supply factor to diffuseinto the at least one intracellular compartment of the oocyte. In anembodiment, introducing at least one exogenous energy supplying factorincludes liposomal introduction by way of endocytosis.

In an embodiment, a method of modifying a female reproductive cellincludes introducing at least one exogenous energy supplying factor intoat least one intracellular compartment of a female reproductive cell. Inan embodiment, the female reproductive cell includes at least one of anoogonium, primary oocyte, secondary oocyte, ootid, ovum, polar body,follicular cell, or cumulus cell.

In an embodiment, a method of modifying a reproductive cell includesintroducing at least one exogenous constituent into at least oneintracellular compartment of a reproductive cell, the exogenousconstituent configured to increase at least one endogenous energysupplying factor.

In an embodiment, a method of selecting at least one reproductive cellincludes detecting at least one endogenous energy supplying factor in atleast one reproductive cell, and scoring the at least one reproductivecell based on the detection. In an embodiment, detecting includesmeasuring the amount of at least endogenous energy supplying factor inat least one reproductive cell, and scoring the at least onereproductive cell based on the measurement. In an embodiment, the methodof modifying at least one reproductive cell includes selecting at leastone reproductive cell based on the score.

In an embodiment, the method of modifying at least one reproductive cellincludes manipulating the at least one reproductive cell selected. In anembodiment, manipulating the at least one reproductive cell includesutilizing the at least one reproductive cell in a fertilization event.In an embodiment, manipulating the at least one reproductive cellincludes freezing the at least one reproductive cell. In an embodiment,manipulating the at least one reproductive cell includes geneticallymodifying the at least one reproductive cell. In an embodiment,manipulating the at least one reproductive cell includes supplyingadditional exogenous energy supplying factors, or other agents to the atleast one reproductive cell. In an embodiment, manipulating the at leastone reproductive cell includes stripping the cell membrane of the atleast one reproductive cell.

In an embodiment, a modified reproductive cell is produced by theprocess of introducing at least one exogenous energy supplying factorinto at least one intracellular compartment of the reproductive cell. Inan embodiment, the introducing includes actively or passivelyintroducing the at least one exogenous energy supplying factor. In anembodiment, the reproductive cell has not yet completed meiosis. In anembodiment, the reproductive cell has not yet entered meiosis. In anembodiment, the reproductive cell has not yet completed mitosis. In anembodiment, the reproductive cell has not yet entered mitosis. In anembodiment, the reproductive cell has not yet completed phase I ofmeiosis.

In an embodiment, the reproductive cell is implantable ortransplantable. In an embodiment, the reproductive cell is implanted ortransplanted into at least one subject. In an embodiment, thereproductive cell is implanted or transplanted into at least one subjectsubsequent to modification. In an embodiment, the reproductive cell isimplanted subsequent to modification, into the original subject fromwhich it was extracted.

In an embodiment, the reproductive cell includes, but is not limited to,at least one of a germ cell, gametegonium, gamete, or reproductivesupporting cell. In an embodiment, the reproductive cell includes but isnot limited to at least one oogonium, primary oocyte, secondary oocyte,ootid, ovum, polar body, follicular cell, granulosa cell, cumulus cell,spermatogonia, primary spermatocyte, secondary spermatocyte, spermatid,spermatozoa, Sertoli cell, or Leydig cell.

In an embodiment, an increase in intracellular ATP-level is sufficientto remedy the impairment of oocyte mitotic spindle, or impairment ofsperm cell motility. In an embodiment, the energy supplying factor(e.g., ATP) is provided to a germ cell, gamete, or supporting cell inorder to enhance intracellular energy levels (e.g., ATP levels), orcompensate for energy level deficiency (e.g., ATP deficiency). In anembodiment, ATP serves as the energy supplying factor, and is providedto a developing gamete, such as an oocyte, prior to or during oocytemeosis phase I.

In an embodiment, the energy supplying factor (e.g., creatine) issupplied by either general administration or specific, localizedadministration. In an embodiment, the energy supplying factor (e.g.,creatine) is supplied by at least one of peroral delivery, oraldelivery, topical delivery, transdermal delivery, epidermal delivery,intravitreal delivery, transmucosal delivery, inhalation, surgicaldelivery, or injection delivery.

In an embodiment, a modified reproductive cell includes at least oneexogenous energy supplying factor. In an embodiment, the exogenousenergy supplying factor is delivered to the cytoplasm of the modifiedreproductive cell. In an embodiment, the exogenous energy supplyingfactor is delivered to at least one mitochondrion of the modifiedreproductive cell.

In an embodiment, the at least one exogenous energy supplying factorincludes at least one of exogenous creatine, exogenous phosphocreatine,exogenous creatine kinase, exogenous chlorophyll, exogenous lactate,exogenous glucose or other carbohydrate, exogenous calcium or othermineral, exogenous ATP, exogenous ADP, exogenous AMP, or a precursorthereof.

In an embodiment, at least one reproductive cell is selected formodification based on one or more reproductive cell parameters. In anembodiment, the one or more reproductive cell parameters relate to atleast one property of the reproductive cell. In an embodiment, the oneor more reproductive cell parameters include at least one ofreproductive cell size, reproductive cell stage, reproductive cellquality, health of the subject from which the reproductive celloriginates, species of the subject from which the reproductive celloriginates, cleavage rate of the reproductive cell, metabolic profile ofthe reproductive cell, genomic profile of the reproductive cell,transcriptomic profile of the reproductive cell, proteomic profile ofthe reproductive cell, or storage conditions of the reproductive cell(if previously stored ex vivo). In an embodiment, the storage conditionsof the reproductive cell include at least one of duration of storagetime, storage temperature, storage size, reproductive cell dilution, orstorage solution(s).

In an embodiment, the at least one subject includes at least one of aplant, alga, or animal. In an embodiment, the at least one subjectincludes at least one of a vertebrate or invertebrate. In an embodiment,the at least one subject includes at least one of an amphibian, mammal,reptile, fish, or bird. In an embodiment, the at least one subjectincludes at least one human. In an embodiment, the at least one plantincludes at least one of a food crop, ornamental, aquatic plant. In anembodiment, the at least one plant includes at least one floweringplant, herb, shrub, bush, tree, or vegetable.

In an embodiment, a method includes detecting at least one reproductivecell indicator. In an embodiment, the at least one reproductive cellindicator includes at least one of a property of the reproductive cell(such as a reproductive cell parameter as discussed herein); a propertyof administering the at least one energy supplying factor to thereproductive cell; a property of the modified reproductive cell; aproperty of administering at least one additional round of an energysupplying factor to a modified reproductive cell; reproductive cell ortissue apoptosis; reproductive cell division; reproductive cellcytoskeletal rearrangement; reproductive cell mitochondrial quality,quantity, or arrangement; reproductive cell fertilization success orfailure; or reproductive cell or tissue secretion.

In an embodiment, the detection material includes at least one of aradioactive, luminescent, colorimetric fluorescent or odorous substance.In an embodiment, the at least one detection material includes at leastone of a taggant, contrast agent, sensor, or electronic identificationdevice. In an embodiment, the at least one electronic identificationdevice includes at least one radio frequency identification device. Inan embodiment, the at least one sensor receives information associatedwith at least one of temperature, pH, inflammation, presence of at leastone substance, or biological response to administration of thecomposition. In an embodiment, the at least one detection materialincludes at least one of a diamagnetic particle, ferromagnetic particle,paramagnetic particle, super paramagnetic particle, particle withaltered isotope, or other magnetic particle.

In an embodiment, a method of modifying a reproductive cell includesdelivering at least one exogenous energy supplying factor to theintracellular compartment of a reproductive cell. In an embodiment, theenergy supplying factor (e.g., ATP) is injected, infused, diffused,electroporated, or otherwise delivered to the intracellular compartmentof a reproductive cell.

In an embodiment, the method further includes detecting at least oneendogenous energy supplying factor in the reproductive cell prior todelivering the at least one exogenous energy supplying factor thereto.In an embodiment, the method further includes measuring at least oneendogenous energy supplying factor in the reproductive cell prior todelivering the at least one exogenous energy supplying factor thereto.In an embodiment, the at least one exogenous energy supplying factor isdelivered in response to the presence or level of at least oneendogenous energy supplying factor in the reproductive cell, thusregulating the overall level of the energy supplying factor in thereproductive cell.

In an embodiment, the method further includes detecting or measuring thelevel of the at least one exogenous energy supplying factor in thereproductive cell subsequent to delivery thereto. In an embodiment, theat least one exogenous energy supplying factor is provided in responseto the presence or level of at least one exogenous energy supplyingfactor in the reproductive cell. In an embodiment, the at least oneexogenous energy supplying factor is provided in response to thepresence or level of at least one reproductive cell indicator, asdescribed herein.

In an embodiment, delivering at least one exogenous energy supplyingfactor includes injecting the reproductive cell with at least oneexogenous energy supplying factor, for example, by intracytoplasmicinjection. In an embodiment, delivering at least one exogenous energysupplying factor to the intracellular compartment of a reproductive cellincludes providing the at least one exogenous energy supplying factor tothe intracellular mitochondria of the reproductive cell.

Oocyte microinjection, including intracytoplasmic sperm injection (ICSI)involves precise maneuver of the oocyte for injection of an agent (e.g.,in the case of ICSI, the oocyte is injected directly with a single livesperm cell for fertilization). As discussed herein, particularly in theExamples section, the procedure includes a female animal subject who hasundergone ovarian stimulation with fertility medications that result inmaturation of one or more oocytes. The oocytes are aspirated, forexample by utilizing vaginal ultrasound for guidance, and incubated. Thesemen sample is typically prepared by centrifuging in medium in order toseparate live sperm cells from debris and most of the dead sperm cells.A single live sperm cell is then isolated from the resultant live spermfraction, and is injected into the oocyte.

Moreover, electroporation is reportedly used for successful genetransfer into spermatozoa cells of various species of fish. See, e.g.,Tsai, Mol. Rep. and Dev. vol. 56, pp. 281-284 (2000), which isincorporated herein by reference.

In an embodiment, a method of maintaining or restoring mitochondrialfunction in a reproductive cell, or reproductive cell vigor, includesadministering to a subject containing at least one reproductive cell, asufficient amount of at least one exogenous energy supplying factor, orat least one agent that promotes endogenous production or retention ofat least one endogenous energy supplying factor. Likewise, in anembodiment, a method of maintaining or restoring reproductive cell vigorin a subject includes administering to the subject containing at leastone reproductive cell, a sufficient amount of at least one exogenousenergy supplying factor, or at least one agent that promotes endogenousproduction or retention of at least one endogenous energy supplyingfactor. In an embodiment, mitochondrial function is assessed, forexample, by measuring the mitochondrial membrane potential (Δψ_(m)).Methods to measure mitochondrial membrane potential are known in theart, and are described in detail herein at the Examples section.

Administering angiotensin receptor inhibitor or angiotensin convertingenzyme inhibitor to human subjects reportedly results in an increase inblood creatine levels. See, e.g., Palmer, Nephrol. Dial. Transplant.Vol. 18, pp. 1973-1975 (2003), which is incorporated herein byreference.

In an embodiment, a method of maintaining or restoring reproductive cellvigor includes administering to a subject containing at least onereproductive cell, an angiotensin receptor blocker (ARB) or anangiotensin converting enzyme inhibitor (ACE-I) sufficient to increasethe endogenous ATP level of at least one of the reproductive cells. Inan embodiment, the angiotensin receptor blocker includes angiotensin-IIreceptor blocker.

In an embodiment, the intracellular levels of ATP are increased in asubject by way of providing creatine supplementation to the subject. Forexample, creatine monohydrate has been reported to provide benefits whenconsumed by human adults. See, e.g., Brose, et al., J. Gerontaol. A.Biol. Sci. Med. Sci., vol. 58, no. 1, pp. 11-9 (2003), which isincorporated herein by reference. In an embodiment, creatinesupplementation is administered to a subject containing at least onereproductive cell, in a sufficient amount to increase the intracellularlevel of ATP in at least one reproductive cell of the subject.

In an embodiment, for example, creatine monohydrate is provided to ahuman subject as at least 1 gram/day, at least 2 grams/day, at least 3grams/day, at least 4 grams/day, at least 5 grams/day, at least 6grams/day, at least 7 grams/day, at least 8 grams/day, at least 9grams/day, at least 10 grams/day, at least 11 grams/day, at least 12grams/day, at least 13 grams/day, at least 14 grams/day, at least 15grams/day, at least 16 grams/day, at least 17 grams/day, at least 18grams/day, at least 19 grams/day, at least 20 grams/day, at least 21grams/day, at least 22 grams/day, at least 23 grams/day, at least 24grams/day, at least 25 grams/day, at least 26 grams/day, at least 27grams/day, at least 28 grams/day, at least 29 grams/day, at least 30grams/day, at least 31 grams/day, at least 32 grams/day, at least 33grams/day, at least 34 grams/day, at least 35 grams/day, at least 36grams/day, at least 37 grams/day, at least 38 grams/day, at least 39grams/day, at least 40 grams/day, or any value less than ortherebetween, for a duration of approximately 1 day, approximately 2days, approximately 3 days, approximately 4 days, approximately 5 days,approximately 6 days, approximately 7 days, approximately 8 days,approximately 9 days, approximately 10 days, approximately 11 days,approximately 12 days, approximately 13 days, approximately 14 days,approximately 3 weeks, approximately 4 weeks, approximately 5 weeks,approximately 6 weeks, approximately 7 weeks, approximately 8 weeks,approximately 9 weeks, approximately 10 weeks, approximately 11 weeks,approximately 12 weeks, approximately 13 weeks, approximately 14 weeks,approximately 15 weeks, approximately 16 weeks, approximately 17 weeks,approximately 18 weeks, approximately 19 weeks, approximately 20 weeks,or any value therebetween. In an embodiment, the creatinesupplementation continues until a successful pregnancy develops. In anembodiment, the creation supplementation continues until a successfulharvest of at least one reproductive cell is completed.

Creatine supplementation can be provided to the subject by any standardmethod, including oral delivery, peroral delivery, injection, topicaldelivery, transdermal delivery, epidermal delivery, intravitrealdelivery, transmucosal delivery, inhalation, or surgical delivery. Thecreatine supplementation is formulated according to the route ofadministration.

In an embodiment, an implantable delivery device comprises: a housingincluding at least one reservoir containing a composition including atleast one exogenous energy supplying factor; the reservoir configured toreceive, retain, and dispense at least a portion of the composition toat least one reproductive cell or tissue; and at least one componentconfigured to administer the composition to the at least onereproductive cell or tissue.

Kits

In an embodiment, kits are included for any of the various aspectsdisclosed herein. For example, in an embodiment, a kit includes adetection material responsive to at least one reproductive cellindicator, and means for administering at least one exogenous energysupplying factor to at least one reproductive cell. In an embodiment,the kit includes the at least one exogenous energy supplying factor. Inan embodiment, the kit includes a delivery device. In an embodiment, thekit includes at least one tool for selecting at least one reproductivecell for manipulation. In an embodiment, a kit includes standardpackaging or instructions for use.

As indicated in the Figures, FIG. 1 illustrates 100 a delivery device,comprising: 110 a housing including at least one reservoir containing atleast one composition including at least one exogenous energy supplyingfactor; the reservoir configured to receive, retain, and dispense atleast a portion of the composition to at least one reproductive cell ortissue. In an embodiment, 115, the delivery device is implantable. In anembodiment, 120, wherein the at least one composition further includesat least one pharmaceutically-acceptable carrier or excipient.

In an embodiment 130, the housing includes one or more ports. In anembodiment 140, at least one of the one or more ports includes at leastone outlet port or at least one inlet port. In an embodiment 150, thedevice further comprises one or more controllable output mechanismsoperably linked to the one or more ports to control dispensing of atleast a portion of the composition from the at least one reservoir. Inan embodiment 155, the at least one controllable output mechanismincludes at least one of a micropump, valve, or actuator. In anembodiment 160, the valve includes at least one of a one-way valve, orpressure settable valve. In an embodiment 170, the actuator includes atleast one of a piezoelectric actuator, electrostatic actuator, thermalactuator, shape-memory alloy actuator, bioactuator, or magneticactuator. In an embodiment 175, the at least one controllable outputmechanism includes at least one thermal or nonthermal gate incommunication with the at least one outlet port of the at least onereservoir.

As illustrated in FIG. 2, in an embodiment 200, the device furthercomprises at least one control circuitry configured to control the atleast one controllable output mechanism. In an embodiment 205, the atleast one control circuitry is configured to generate and transmit anelectromagnetic control signal. In an embodiment 210, the device furthercomprises at least one memory mechanism for storing instructions forgenerating and transmitting the electromagnetic control signal. In anembodiment 220, the at least one control circuitry is configured fortime-release of at least a portion of the at least one composition fromthe at least one reservoir.

In an embodiment 230, the at least one control circuitry is configuredfor variable programming control of the at least one controllable outputmechanism. In an embodiment 240, the at least one control circuitry isconfigured to control the release of the composition. In an embodiment250, the at least one control circuitry is configured to control atleast one of the rate of release, amount of release, or time of releaseof the composition. In an embodiment 260, the at least one controlcircuitry is configured to generate and transmit an electromagneticcontrol signal configured to control the at least one controllableoutput mechanism. In an embodiment 270, the at least one controlcircuitry is configured to control the at least one controllable outputmechanism for time-release of at least a portion of the composition fromthe at least one reservoir. In an embodiment 280, the at least onecontrol circuitry is configured for variable programming control of theat least one controllable output mechanism.

In an embodiment 290, the at least one control circuitry is configuredto control release of the composition or a portion thereof in responseto a signal from a sensor. In an embodiment 300, the signal representsinformation from at least one reproductive cell indicator. In anembodiment 310, the at least one or more reproductive cell indicatorincludes at least one of a property of the reproductive cell, a propertyof administering the at least one energy supplying factor to thereproductive cell; a property of the modified reproductive cell; aproperty of administering at least one additional round of an energysupplying factor to a modified reproductive cell; reproductive cell ortissue apoptosis; reproductive cell division; reproductive cellcytoskeletal rearrangement; reproductive cell mitochondrial quality,quantity, or arrangement; reproductive cell fertilization success orfailure; or reproductive cell or tissue secretion.

As illustrated in FIG. 3, in an embodiment, the device further comprisesat least one transducer. In an embodiment 310, the device furthercomprises at least one receiver. In an embodiment 320, the at least onereceiver is configured to receive information from at least one distalor remote sensor. In an embodiment 330, the receiver is configured toobtain release instructions or authorization to release the composition.In an embodiment 340, the receiver is configured to receive programminginstructions or data for the controller.

In an embodiment 350, the device further comprises at least onetransmitter. In an embodiment 360, the at least one transmitter isconfigured to transmit information regarding one or more of the date,time, presence or approximate quantity of release of the at least onecomposition, or information regarding at least one reproductive cell orsubstance associated with the release of the composition.

In an embodiment 370, the device further comprises at least one powersource. In an embodiment 380, the device further comprises at least onedetection material. In an embodiment 390, the reservoir is configuredfor controlled release of at least one detection material.

As illustrated in FIG. 4, in an embodiment 400, the at least onedetection material includes at least one of a radioactive, luminescent,colorimetric, fluorescent, or odorous substance. In an embodiment 410,the at least one detection material includes at least one of a taggant,contrast agent, or electronic identification device. In an embodiment420, the at least one electronic identification device includes at leastone radio frequency identification device. In an embodiment 430, thedelivery device further comprises at least one sensor. In an embodiment440, the at least one sensor is configured to receive informationassociated with at least one, of temperature, pH, inflammation, presenceof at least one substance, detection material, or biological response toadministration of the composition. In an embodiment 450, the deliverydevice further comprises a controller configured to respond to the atleast one sensor. In an embodiment 460, the at least one detectionmaterial includes at least one of a diamagnetic particle, ferromagneticparticle, paramagnetic particle, super paramagnetic particle, particlewith altered isotope, or other magnetic particle. In an embodiment 470,the at least one detection material is responsive to at least one of thepresence or level of the composition; or the presence or level of atleast one reproductive cell or substance associated with the dispensingof the composition. In an embodiment 480, the at least one detectionmaterial is responsive to the approximate quantity of at least one ofthe composition, or at least one reproductive cell or substanceassociated with the release of the composition.

As illustrated in FIG. 5, the at least one detection material isresponsive to at least one of: enzyme, acid, amino acid, peptide,polypeptide, protein, oligonucleotide, nucleic acid, ribonucleic acid,oligosaccharide, polysaccharide, glycopeptide, glycolipid, lipoprotein,sphingolipid, glycosphingolipid, glycoprotein, peptidoglycan, lipid,carbohydrate, metalloprotein, proteoglycan, chromosome, adhesionmolecule, cytokine, chemokine, immunoglobulin, antibody, antigen,platelet, extracellular matrix, blood plasma, cell wall, hormone,organic compound, inorganic compound, salt, or cell ligand. In anembodiment 510, the at least one detection material is responsive to atleast one of: glucose, lactate, urea, uric acid, glycogen, oxygen,carbon dioxide, carbon monoxide, ketone, nitric oxide, nitrous oxide,alcohol, alkaloid, pH, albumin, ATP, NADH, FADH₂, pyruvate, creatinine,cholesterol, alpha-fetoprotein, chorionic gonadotropin, estrogen,progesterone, testosterone, thyroxine, melatonin, calcitonin,antimullerian hormone, angiotensin, follicle-stimulating hormone,gonadotropin-releasing hormone, mitochondria, growth hormone, growthhormone-releasing hormone, insulin, human placental lactogen, oxytocin,luteinizing hormone, prolactin, cortisol, aldosterone, estradiol,estriol, estrone, leukotriene, vitamin, mineral, renin, DHEA, DHT,alloisoleucine, or a precursor of any thereof.

In an embodiment 520, the device further comprises at least one memorymechanism for storing instructions for generating and transmitting anelectromagnetic control signal. In an embodiment 530, the device furthercomprises at least one imaging apparatus capable of imaging theapproximate quantity within a treatment region relating to at least oneof the composition; at least one reproductive cell, or at least onesubstance associated with dispensing the composition. In an embodiment540, the device further comprises at least one memory location forrecording information related to sensing information, imaginginformation, transmitting information, or other information related tothe delivery device. In an embodiment 550, the at least one memorylocation is configured to record information regarding at least onesensor. In an embodiment 560, the at least one memory location isconfigured to record information regarding at least one of a sensedcondition, history, or performance of the device.

As illustrated in FIG. 6, in an embodiment 600, the at least one memorylocation is configured to record information regarding one or more ofthe date, time, presence or approximate quantity of at least one of theadministered composition; or at least one reproductive cell or substanceassociated with the dispensing of the composition. In an embodiment 610,the at least one reproductive cell or substance associated withdispensing of the composition includes at least one of an organic orinorganic small molecule, nucleic acid, amino acid, peptide,polypeptide, protein, glycopeptide, glycoprotein, glycolipid,lipopolysaccharide, peptidoglycan, proteoglycan, lipid, lipoprotein,glycospingolipid, metalloprotein, liposome, chromosome, nucleus, acid,base, buffer, protic solvent, aprotic solvent, carbohydrate, ATP,creatine, lactate, phosphocreatine, pyruvate, glucose,lipopolysaccharide, vitamin, mineral, cytokine, chemokine, antibody,element, hormone, virus, enzyme, oligonucleotide, ribonucleic acid,oligosaccharide, polysaccharide, adhesion molecule, NADH, FADH₂,estrogen, progesterone, testosterone, thyroxine,corticotrophin-releasing hormone, follicle-stimulating hormone,gonadotropin-releasing hormone, growth hormone, growth hormone-releasinghormone, insulin, oxytocin, luteinizing hormone, leptin, prolactin,cortisol, aldosterone, estradiol, estriol, estrone, DHEA, DHT, germcell, gametegonium, gamete, or reproductive supporting cell. In anembodiment, the reproductive cell includes but is not limited to atleast one oogonium, primary oocyte, secondary oocyte, ootid, ovum, polarbody, follicular cell, granulosa cell, cumulus cell, spermatogonia,primary spermatocyte, secondary spermatocyte, spermatid, spermatozoa,Sertoli cell, or Leydig cell.

In an embodiment 620, the device further comprises at least oneinformation transmission mechanism configured to transmit informationrecorded by the at least one electronic memory location. In anembodiment 630, the device is located in or is substantially in the formof one or more of a spray apparatus, pump apparatus, bioreactor, ordrilling apparatus. In an embodiment 640, the device is substantially inthe form of one or more of a patch, oral inhaler, nasal spray or otherorifice spray, iontophoretic apparatus, oral consumable, ocular or oticdropper or spray, stent, shunt, orifice insert, ductal implant, organimplant, orifice spray or inhaler, sutures, surgical staples, bandages,micro-electromechanical device, nano-electromechanical device, orabsorbable mesh. In an embodiment 650, the device further comprises oneor more inlet mechanisms for receiving external delivery of the at leastone composition.

As illustrated in FIG. 7, in an embodiment 700, a system comprises: 710at last one computing device, 720 at least one delivery deviceconfigured to receive, retain and dispense at least a portion of acomposition including at least one energy supplying factor to at leastone reproductive cell; and a recordable medium including one or moreinstructions that when executed on the computing device cause thecomputing device to regulate dispensing of at least a portion of thecomposition. In an embodiment 730, the at least one delivery deviceincludes at least one implantable delivery device. In an embodiment 740,wherein the at least one computing device includes at least onecomputing device located on or in the at least one delivery device. Inan embodiment 750, the at least one computing device includes at leastone computing device located remotely from the at least one deliverydevice. In an embodiment 760, the at least one computing device includesone or more of a desktop computer, workstation computer, or computingsystem. In an embodiment 770, the at least one computing system includesone or more of a cluster of processors, networked computer, tabletpersonal computer, laptop computer, mobile device, mobile telephone, orpersonal digital assistant computer. In an embodiment 780, the systemfurther comprises one or more instructions that when executed on the atleast one computing device cause the at least one computing device togenerate at least one output to a user.

As illustrated in FIG. 8, in an embodiment 800, the at least one outputincludes at least one graphical illustration of one or more of thecomposition, at least one reproductive cell or substance associated withthe composition, at least one property of the delivery device; or atleast one property of dispensing the at least one delivery device. In anembodiment 810, the at least one output includes at least one protocolfor making the composition. In an embodiment 820, the at least oneoutput includes at least one protocol for administering the compositionto the at least one reproductive cell. In an embodiment 830, the.userincludes at least one entity. In an embodiment 840, the entity includesat least one person, or computer. In an embodiment 850, the outputincludes output to a user readable display. In an embodiment 860, theuser readable display includes a human readable display. In anembodiment 870, the user readable display includes one or more activedisplays. In an embodiment 880, the user readable display includes oneor more passive displays. In an embodiment 890, the user readabledisplay includes one or more of a numeric format, graphical format, oraudio format.

As illustrated in FIG. 9, the system further comprises one or moreinstructions for determining one or more parameters for selecting the atleast one reproductive cell from other reproductive cells. In anembodiment 910, the system further comprises one or more instructionsfor determining one or more parameters for administering the compositionto at least one reproductive cell. In an embodiment 920, the systemfurther comprises one or more instructions for receiving informationrelated to one or more reproductive cell indicators prior to, during, orsubsequent to administering the composition to the at least onereproductive cell. In an embodiment 930, the information related to oneor more reproductive cell indicators includes information from at leastone of an assay, image, or gross assessment of the at least onereproductive cell or reproductive cell related tissue prior to, during,or subsequent to administering the composition. In an embodiment 940,the assay includes at least one technique including spectroscopy,microscopy, electrochemical detection, polynucleotide detection,histological examination, biopsy analysis, fluorescence resonance energytransfer, electron transfer, enzyme assay, electrical conductivity,isoelectric focusing, chromatography, immunoprecipitation,immunoseparation, aptamer binding, filtration, electrophoresis,immunoassay, or radioactive assay.

In an embodiment 950, the at least one image includes one or more imagesacquired by at least one of laser, holography, x-ray crystallography,optical coherence tomography, computer-assisted tomography scan,computed tomography, magnetic resonance imaging, positron-emissiontomography scan, ultrasound, x-ray, electrical-impedance monitoring,microscopy, spectrometry, flow cytommetry, radioisotope imaging, thermalimaging, infrared visualization, multiphoton calcium-imaging,photography, or in silico generation.

As illustrated in FIG. 10, in an embodiment 1000, the at least onereproductive cell is located in at least one of in situ, in vitro, invivo, in utero, in planta, in silico, or ex vivo. In an embodiment 1020,the at least one reproductive cell is located in at least one subject.In an embodiment 1030, the at least one subject includes at least one ofan invertebrate or vertebrate animal. In an embodiment 1040, the atleast one subject includes at least one of a reptile, mammal, amphibian,bird, or fish. In an embodiment 1050, the at least one subject includesat least one human. In an embodiment 1060, the at least one subjectincludes at least one plant.

As illustrated in FIG. 11, in an embodiment 1100, a computer programproduct comprises: 1110 a recordable medium bearing one or moreinstructions for regulating dispensing of at least one delivery deviceto at least one reproductive cell, wherein the delivery device includesa composition including at least one exogenous energy supplying factor,and generating at least one output. In an embodiment 1120, therecordable medium includes a computer-readable medium. In an embodiment1130, the recordable medium includes a communications medium. In anembodiment 1140, the computer program product further comprises one ormore instructions for receiving information related to one or morereproductive cell indicators prior to, during, or subsequent toadministering the composition. In an embodiment 1160, the outputincludes at least one protocol for making the composition. In anembodiment 1170, the output includes at least one protocol foradministering the composition to at least one reproductive cell. In anembodiment 1180, the output includes at least one graphical illustrationof one or more of the composition, or at least one reproductive cell orsubstance associated with the at least one reproductive cell, at leastone property of the at least one delivery device, or at least oneproperty of dispensing the at least one delivery device. In anembodiment 1190, the computer program product further comprises one ormore instructions for displaying results of the processing.

As illustrated in FIG. 12, in an embodiment 1200, a computer-implementedmethod comprises: 1210 regulating dispensing a composition from at leastone delivery device to at least one reproductive cell, the compositionincluding at least one energy supplying factor. In an embodiment 1220,the at least one delivery device includes at least one implantabledelivery device. In an embodiment 1230, the computer-implemented methodfurther comprises generating at least one output. In an embodiment 1240,the generating at least one output includes generating at least oneoutput to a user. In an embodiment 1250, the user includes at least oneentity. In an embodiment 1260, the entity includes at least one person,or computer. In an embodiment 1270, the at least one output includes atleast one graphical illustration of one or more of the composition, orat least one reproductive cell or substance associated with the at leastone reproductive cell; at least one property of the at least onedelivery device; or at least one property of dispensing the at least onedelivery device. In an embodiment 1280, the at least one output includesat least one protocol for making the composition based on one or moreparameters of the at least one reproductive cell. In an embodiment 1285,the at least one output includes one or more parameters for selectingthe at least one reproductive cell from other reproductive cells.

As illustrated in FIG. 13, in an embodiment 1300, the at least oneoutput includes at least one protocol for dispensing the at least onecomposition to the at least one reproductive cell. In an embodiment1302, the dispensing the at least one composition to the at least onereproductive cell includes dispensing the at least one composition inresponse to one or more reproductive cell indicators. In an embodiment1305, the at least one output includes at least one output to a userreadable display. In an embodiment 1308, the user readable displayincludes a human readable display. In an embodiment 1310, the userreadable display includes one or more active displays. In an embodiment1320, the user readable display includes one or more passive displays.In an embodiment 1330, the user readable display includes one or more ofa numeric format, graphical format, or audio format. In an embodiment1340, the computer-implemented method further comprises receivinginformation related to one or more reproductive cell indicators priorto, during, or subsequent to administering the composition to the atleast one reproductive cell. In an embodiment 1350, the receivinginformation related to one or more reproductive cell indicators includesinformation from at least one of an assay, image, or gross assessment ofthe at least one biological tissue prior to, during, or subsequent. toadministering the composition.

As illustrated in FIG. 14, in an embodiment 1400, the assay includes atleast one technique including spectroscopy, microscopy, electrochemicaldetection, polynucleotide detection, histological examination, biopsyanalysis, fluorescence resonance energy transfer, electron transfer,enzyme assay, electrical conductivity, isoelectric focusing,chromatography, immunoprecipitation, immunoseparation, aptamer binding,filtration, electrophoresis, immunoassay, or radioactive assay. In anembodiment 1420, the at least one image includes one or more imagesacquired by at least one of laser, holography, x-ray crystallography,optical coherence tomography, computer-assisted tomography scan,computed tomography, magnetic resonance imaging, positron-emissiontomography scan, ultrasound, x-ray, electrical-impedance monitoring,microscopy, spectrometry, flow cytommetry, radioisotope imaging, thermalimaging, infrared visualization, multiphoton calcium-imaging,photography, or in silico generation. In an embodiment 1440, the atleast one reproductive cell is located in at least one of in situ, invitro, in vivo, in utero, in planta, in silico, or ex vivo. In anembodiment 1450, the at least one reproductive cell is at leastpartially located in at least one subject. In an embodiment 1460, the atleast one subject includes at least one of an invertebrate or vertebrateanimal. In an embodiment 1470, the at least one subject includes atleast one of a reptile, mammal, amphibian, bird, or fish. In anembodiment 1480, the at least one subject includes at least one human.In an embodiment 1490, the at least one subject includes at least oneplant.

PROPHETIC EXAMPLES Example 1 Method to Provide ATP to Oocytes from aFemale Subject of Advanced Age Undergoing in vitro Fertilization

A female subject who is at an advanced maternal age, 43 years old,undergoes ovarian stimulation to induce maturation of multiple folliclesand produce oocytes, which are harvested by transvaginal oocyteretrieval. The harvested oocytes are tested with a mitochondrialmembrane potential assay to assess their overall health. Based on theassay results, ATP encapsulated in liposomes is delivered to thecytoplasm of the harvested oocytes in need thereof. The modified oocytesare reanalyzed to assess oocyte quality prior to selection for in vitrofertilization (IVF) and cryopreservation.

Methods to stimulate multiple ovarian follicles and isolate multipleoocytes are known in the art (see e.g., Rossato et al., Hum. Reprod. 14:694-697, 1999 which is incorporated herein by reference). Ovarianstimulation is accomplished by administering a gonadotrophin releasinghormone analog (e.g., triptorelin, Trelstar La available from WatsonPharma, Inc., Corona, Calif.), and follicle stimulating hormone (FSH;e.g., Follistim® AQ available from Schering Plough, Kenilworth, N.J.).FSH treatments are given for approximately 9 days and the follicles aremonitored by vaginal ultrasound until the follicles reach a diametergreater than about 18 mm. Then human chorionic gonadotrophin (HCG,10,000 IU; e.g., Pregnyl®, available from Schering Plough, Kenilworth,N.J.) is administered. Approximately 36 hours after HCG administrationthe oocytes are retrieved by transvaginal aspiration. Procedures anddevices for transvaginal ultrasonography and aspiration of ovarianfollicles are known in the art (see e.g., Wang and Sauer, Therap. andClinical Risk Mgmt, 2: 355-364, 2006 which is incorporated herein byreference). The retrieved oocytes are tested to assess mitochondrialhealth.

Individual oocytes are tested for their oxygen respiration rate using areal-time, noninvasive system (see e.g., Scott et al., Rep. Biomed.Online 17: 461-469, 2008, which is incorporated herein by reference).Oocytes are classified microscopically as immature (germinal vesicle andmetaphase I oocytes) or metaphase II oocytes. Oocytes are washed free ofall cannulus and granulosa cells and placed in individual wells of aglass tray in Sage Complete Embryo Culture medium (available from SageIn vitro Fertilization, Inc., Trumbull, Conn.). Oxygen respiration ratemeasurements are made with microelectrodes that measure the steady-statediffusive oxygen flux toward oocytes placed in the bottom of narrowwells in culture trays. An instrument for measuring respiration rates inoocytes, EmbryoScope™, is available from Unisense Fertilitech, Aarhus,Denmark. A baseline respiration rate (BRR) is calculated from repeatedmeasurement of the oxygen concentration gradients in the well over aninitial 5 hour time period. The mean BRR for oocytes from mothersgreater than 40 years old is significantly lower (P<0.01) than that formothers less than 35 years of age (BRR=0.461 nL O₂/hr versus BRR=0.575nL O₂/hr respectively (see e.g., Scott et al., Ibid.) In addition, alloocytes are tested to determine their mitochondrial membrane potentials.

Mitochondrial membrane potential (Δψ) is a key indicator ofmitochondrial health and metabolic activity. Methods to measure Δψ inlive cells are known in the art (see e.g., Distelmaier et al., CytometryPart A, 73A: 129-138, 2008, which is incorporated herein by reference).To measure Δψ_(m), oocytes are incubated with a fluorescent cationicdye, tetramethyl rhodamine methyl ester (TMRM; available from LifeTechnologies Corp., Carlsbad, Calif.), that is taken up and sequesteredby mitochondria. Oocytes are incubated in Medium 199 (available fromLife Technologies Corp., Carlsbad, Calif.) containing 100 nM TMRM forabout 25 minutes at 37° C., and then washed with phosphate bufferedsaline (PBS). To measure TMRM fluorescence, an inverted microscope(available from Carl Zeiss Microimaging, Inc., Thornwood, N.Y.) with anX63 oil immersion objective is used and the oocytes are maintained at37° C. in a heated chamber. TMRM sequestered within mitochondria isexcited with 540 nm wavelength light for approximately 100 milliseconds,and fluorescent images emitted at approximately 565 nm are captured on aCoo1SNAP HQ monochrome CCD-camera (available from Roper ScientificPhotometrics, Vianen, The Netherlands). Image processing and analysisare performed using Image Pro Plus 5.1 software (available from MediaCybernetics, Silver Spring, Md.) and Origin Pro 7.5 software (availablefrom Originlabs, Northampton, Mass.). Small changes in Δψ_(m) (e.g.,approximately 12 millivolts) are seen as a reduction in averagefluorescence intensity from the mitochondria in an oocyte. A mean Δψ_(m)of approximately −119 millivolts is seen for healthy normal cells. See,e.g., Distelmaier et al., Ibid.)

Oocytes with diminished Δψ_(m)are cultured with ATP encapsulated inliposomes to supplement cytoplasmic ATP. Oocytes are cultured in mediacomprised of follicular fluid (obtained during follicle aspiration) anda balanced salt solution, e.g., Ham's F10 media. Liposomes containing36-38 mole percent of ATP ((moles of ATP/moles of lipid)×100) are addedto the cultures to deliver approximately 2 pmole of ATP to the cytoplasmof each oocyte to promote oocyte health (see e.g., Van Blerkom et al.,Hum. Reprod. 10: 415-424, 1995 which is incorporated herein byreference). Methods to prepare liposomes containing ATP are known in theart (see e.g., Liang et al., J. Microencapsulation, 21: 251-261, 2004;and U.S. Pat. No. 7,056,529; each of which is incorporated herein byreference).

Liposomes comprised of phosphatidyl choline and cholesterol are combinedwith a 400 mM ATP solution and subjected to a freeze thaw process, andthen purified (see e.g., Liang et al., Ibid.; lipids and ATP areavailable from Sigma-Aldrich, St. Louis, Mo.). Alternatively liposomesloaded with ATP are commercially available from FormuMax ScientificInc., Palo Alto, Calif. Oocytes are cultured at about 37° C. withATP-loaded liposomes in approximately 5% CO₂ and 95% air for about 4-8hours (see e.g., U.S. Pat. No. 5,563,059, which is incorporated hereinby reference). Oocyte quality is assessed by measuring Δψ, and bymicroscopic inspection (see e.g., Van Blerkom et al., Ibid.) which isincorporated herein by reference).

Normal appearing oocytes with a normal distribution of cytoplasmicorganelles and a Δψ_(m) of −119 millivolts are selected for IVF andcryopreservation. Selected oocytes are fertilized in vitro withapproximately 100,000 spermatozoa per oocyte. Methods to collect andprepare spermatozoa and to inseminate oocytes are known in the art (seee.g., Rossato et al., Ibid.). Fertilization is evaluated microscopicallyafter 16-20 hours by identification of pronuclei, and 46-48 hours afterinsemination the embryos are classified by the number and shape ofblastomeres and the percentage of fragments. Alternatively, healthyoocytes with a Δψ_(m) of approximately −119 millivolts are frozen forfuture use. Germinal vesicle, metaphase I and metaphase II oocytes arefrozen using a slow freezing method (see e.g., Tao and Del Valle, J.Assist. Reprod. Genet. 25: 287-296, 2008 which is incorporated herein byreference). Oocytes are treated with a cryoprotectant (e.g., dimethylsulfoxide available from Sigma-Aldrich, St. Louis, Mo.) and step-wisecooled to −7° C. at −1° C./min; then cooled to −35° C. at −0.3° C./min;then cooled rapidly to −80° C. and ultimately plunged into liquidnitrogen (at −196° C.). Each oocyte is stored under liquid nitrogen in amicroculture dish with its individual well location referenced to acomputer address. Information stored on the computer includes: theoocyte's microscopic evaluation, respiration rate, Δψ_(m), and themother's identity, medical history, and genetic information.

Example 2 Implanted Device to Supply ATP by Microinjection to OocytesWithin Ovarian Follicles and Improve the Quality of Oocytes that areSelected for in vitro Fertilization

A female subject of advanced maternal age, 40 years of age, is treatedwith fertility drugs to stimulate follicle development in her ovaries.Follicle development is stimulated by administering a gonadotrophinreleasing hormone analog (e.g., triptorelin, Trelstar La available fromWatson Pharma, Inc., Corona, Calif.) and follicle stimulating hormone(FSH; e.g., Follistim® AQ available from Schering Plough, Kenilworth,N.J.). FSH treatments are given for approximately 9 days and thefollicles are monitored by vaginal ultrasound until the follicles reacha diameter of approximately 12 mm.

Guided by ultrasound imaging each follicle of approximately 12 mm indiameter is implanted with a micro-electromechanical system (MEMS)device. The MEMS device contains a sensor for an oocyte indicator, areservoir, a nanopump to deliver liposomal ATP, and a Radio FrequencyIdentification (RFID) tag. The MEMS sensor detects oxygen in follicularfluid, an indicator of oocyte health, and delivers liposomes containingATP to oocytes in follicles with low oxygen levels (see e.g., U.S.Patent Application No. 2008/0091119, which is incorporated herein byreference).

The oocyte indicator is monitored twice daily by the MEMS device andadditional doses of liposomal ATP are delivered as required. The MEMSdevice contains a RFID tag that identifies the follicle and links dataon oxygen levels and liposomal ATP delivered to a specific follicle. ARFID reader and remote computing device store and evaluate thecumulative data for each follicle and its associated oocyte.

Micro-electromechanical system devices are injected into ovarianfollicles to sense an indicator of oocyte health, and to deliverliposomal ATP. A MEMS device is constructed with an oxygen sensor toassess the O₂ content of follicular fluid. For example, an implantedmicrosensor that detects oxygen levels in the range of 0-50 mm Hg andtransmits data to an external receiver is known in the art (see e.g.,U.S. Pat. No. 7,010,340, which is incorporated herein by reference). Awireless, implantable MEMS device (see e.g., Product Sheet fromIntegrated Sensing Systems, Ypsilanti, Mich., which is incorporatedherein by reference) is transvaginally injected into each follicleguided by ultrasound imaging, and the oxygen concentration in individualfollicles is transmitted wirelessly to a computing device. The oxygenconcentration for each follicle is measured twice daily, and the dataare stored and analyzed on a computing device. Circuitry on the MEMSdevice detects electronic signals from the oxygen sensor correspondingto suboptimal oxygen concentrations (e.g., less than 50 mm Hg) andactuates a nanopump to deliver liposomal ATP contained in a reservoir onthe MEMS device. The nanopump delivers a liposomal

ATP suspension at a flow rate of approximately 50 milliliters/(min.cm²)to the follicular fluid and the oocyte. (See e.g., U.S. Pat. No.7,540,717, which is incorporated herein by reference). Liposomescontaining 36-38 mole percent of ATP ((moles of ATP/moles of lipid)×100)are administered to deliver approximately 2 pmole of ATP to thecytoplasm of the oocyte. Methods to prepare liposomes containing ATP areknown in the art (see e.g., Liang et al., J. Microencap., 21: 251-261,2004; and U.S. Pat. No. 7,056,529, each of which are incorporated hereinby reference). Liposomes comprised of phosphatidyl choline andcholesterol are combined with a 400 mM ATP solution and subjected to afreeze thaw process and then purified (see e.g., Liang et al., Ibid.;lipids and ATP are available from Sigma-Aldrich, St. Louis, Mo.).Alternatively, liposomes loaded with ATP are commercially available fromFormuMax Scientific Inc., Palo Alto, Calif.

Follicular development is monitored using vaginal ultrasonography andoocytes are retrieved by aspiration of the follicles. When follicles areapproximately 18 mm in diameter human chorionic gonadotrophin (HCG,10,000 IU; e.g., Pregnyl®, available from Schering Plough, Kenilworth,N.J.) is administered and approximately 36 hours after HCGadministration the oocytes are retrieved by transvaginal aspiration.Procedures and devices for transvaginal ultrasonography and aspirationof ovarian follicles are known in the art (see e.g., Wang and Sauer,Therap. and Clin. Risk Mgmt., 2: 355-364, 2006, which is incorporatedherein by reference). Follicular fluid containing an oocyte and the MEMdevice with a micro-RFID tag (see e.g., Product Sheet from HitachiEurope, Ltd., Maidenhead, Berkshire, U.K., which is incorporated hereinby reference) is cultured in vitro prior to in vitro fertilization ofthe oocyte. For example, an RFID tag, such as the Hitatchi Mu-chip, is apassive tag that requires no battery, and is manufactured to enable thechip to operate in heat and moisture (Product Sheet Hitachi RFIDSolutions, Ibid.) A RFID reader (available from Hitachi Europe, Ltd.,Maidenhead, Berkshire, U.K.) is used to detect the MEMS device. The RFIDreader is also used to link the follicle with the corresponding oocyteand the accumulated data on oxygen levels, as well as the amount ofliposomal ATP delivered. Moreover, the RFID tag is used to identify thefemale subject who produced the oocyte as well as the associated data onher medical history, genetics, and the dose and schedule of herfertility drugs.

Retrieved oocytes are tested in vitro in order to select the healthiestoocytes for in vitro fertilization and cryopreservation. As mentioned,the mitochondrial membrane potential (Δψ_(m)) is a key indicator ofmitochondrial health and metabolic activity. Methods to measure Δψ inlive cells are known in the art (see e.g., Distelmaier et al., CytometryPart A, 73A: 129-138, 2008, which is incorporated herein by reference).To measure Δψ_(m), oocytes are incubated with a fluorescent cationicdye, tetramethyl rhodamine methyl ester (TMRM; available from LifeTechnologies Corp., Carlsbad, Calif.), that is taken up and sequesteredby mitochondria. Oocytes are incubated in Medium 199 (available fromLife Technologies Corp., Carlsbad, Calif.) containing 100 nM TMRM forabout 25 minutes at 37° C., and then washed with phosphate bufferedsaline (PBS). In order to measure TMRM fluorescence, an invertedmicroscope (available from Carl Zeiss Microimaging, Inc., Thornwood,N.Y.) with an X63 oil immersion objective is used, and the oocytes aremaintained at 37° C. in a heated chamber. TMRM sequestered withinmitochondria is excited with 540 nm wavelength light for approximately100 milliseconds and fluorescent images emitted at approximately 565 nmare captured on a Coo1SNAP HQ monochrome CCD-camera (available fromRoper Scientific Photometrics, Vianen, The Netherlands). Imageprocessing and analysis are performed using Image Pro Plus 5.1 software(available from Media Cybernetics, Silver Spring, Md.) and Origin Pro7.5 software (available from Originlabs, Northampton, Mass.). Smallchanges in Δψ_(m) (e.g., approximately 12 millivolts) are seen as areduction in average fluorescence intensity from the mitochondria in anoocyte. A mean Δψ_(m) of approximately −119 millivolts is seen forhealthy normal cells (see e.g., Distelmaier et al., Ibid). Oocytes witha mean Δψ_(m) of approximately −119 millivolts and a heterogeneousdistribution of mitochondria clustered around the nuclear membrane(characteristic of oocytes in metaphase I or II) are selected for invitro fertilization and cryopreservation (see e.g., Wang et al., J. ofZhejiang Univ. Sci. B 10: 483-492 (2009) which is incorporated herein byreference).

Selected oocytes are fertilized in vitro with approximately 100,000spermatozoa per oocyte. Methods to collect and prepare spermatozoa andto inseminate oocytes are known in the art (see e.g., Rossato et al.,Ibid.). Fertilization is evaluated microscopically after 16-20 hours byidentification of pronuclei, and 46-48 hours after insemination theembryos are classified for the number and shape of blastomeres and thepercentage of fragments. Alternatively, healthy oocytes with a Δψ_(m) ofapproximately −119 millivolts are frozen for future use. Germinalvesicle, metaphase I oocytes, and metaphase II oocytes are frozen usinga slow freezing method (see e.g., Tao and Del Valle, J. Assist. Reprod.Genet. 25: 287-296, 2008, which is incorporated herein by reference).Oocytes are treated with a cryoprotectant (e.g., dimethyl sulfoxideavailable from Sigma-Aldrich, St. Louis, Mo.) and then cooled to −7° C.at a rate of −1° C./min; then cooled to −35° C. at −0.3° C./min; thencooled rapidly to −80° C. and ultimately plunged into liquid nitrogen(at −196° C.). Each oocyte is stored under liquid nitrogen in amicroculture dish with its MEMS device and RFID tag identifying theoocyte and linking it to information stored on a computer that includes:the oocyte's microscopic evaluation, oxygen levels, liposomal ATPdelivered, Δψ_(m), and the subject's identity, medical history, andgenetic information.

Example 3 A Method to Provide Phosphocreatine to Oocytes in vitro toImprove the Quality and Quantity of Oocytes Suitable for in vitroFertilization and Cryopreservation

A female subject who is at an advanced maternal age undergoes ovarianstimulation to induce multiple follicles and oocytes which are harvestedby ultrasound-guided transvaginal oocyte retrieval. The oocytes aretested with a mitochondrial membrane potential (Δψ_(m)) assay to assesstheir health. Based on the assay results, phosphocreatine is deliveredto the cytoplasm of the oocytes as needed. Following in vitromaturation, the modified oocytes are reanalyzed to assess oocyte qualityprior to selection for in vitro fertilization (IVF) andcryopreservation.

Methods to stimulate multiple ovarian follicles and isolate multipleoocytes are known in the art (see e.g., Rossato et al., Hum. Reprod. 14:694-697, 1999 which is incorporated herein by reference). Ovarianstimulation is accomplished by administering a gonadotrophin releasinghormone analog (e.g., triptorelin, Trelstar La available from WatsonPharma, Inc., Corona, Calif.) and follicle stimulating hormone (FSH;e.g., Follistim® AQ available from Schering Plough, Kenilworth, N.J.).FSH treatments are given for approximately 9 days and the follicles aremonitored by vaginal ultrasound until the follicles reach a diametergreater than about 18 mm.

Next, human chorionic gonadotrophin (HCG, 10,000 IU; e.g., Pregnyl®,available from Schering Plough, Kenilworth, N.J.) is administered andapproximately 36 hours after HCG administration, the oocytes areretrieved by transvaginal aspiration. Procedures and devices fortransvaginal ultrasonography and aspiration of ovarian follicles areknown in the art (see e.g., Wang and Sauer, Therap. and Clin. Risk Mgt.,2: 355-364 (2006), which is incorporated herein by reference). Theretrieved oocytes are tested to assess mitochondrial health.

The mitochondrial membrane potential (Δψ_(m)) is a key indicator ofmitochondrial health and metabolic activity. Methods to measure Δψ_(m)in live cells are known in the art (see e.g., Distelmaier et al.,Cytometry Part A, 73A: 129-138 (2008), which is incorporated herein byreference). To measure Δψ_(m), oocytes are incubated with a fluorescentcationic dye, tetramethyl rhodamine methyl ester (TMRM; available fromLife Technologies Corp., Carlsbad, Calif.), that is taken up andsequestered by mitochondria. Oocytes are incubated in Medium 199(available from Life Technologies Corp., Carlsbad, Calif.) containing100 nM TMRM for about 25 minutes at 37° C., and then washed withphosphate buffered saline (PBS). To measure TMRM fluorescence, aninverted microscope (available from Carl Zeiss Microimaging, Inc.,Thornwood, N.Y.) with an X63 oil immersion objective is used, and theoocytes are maintained at 37° C. in a heated chamber. TMRM sequesteredwithin mitochondria is excited with 540 nm wavelength light forapproximately 100 milliseconds, and fluorescent images emitted atapproximately 565 nm are captured on a CoolSNAP HQ monochrome CCD-camera(available from Roper Scientific Photometrics, Vianen, The Netherlands).Image processing and analysis are performed using Image Pro Plus 5.1software (available from Media Cybernetics, Silver Spring, Md.) andOrigin Pro 7.5 software (available from Originlabs, Northampton, Mass.).Small changes in Δψ_(m) (e.g., approximately 12 millivolts) are seen asa reduction in average fluorescence intensity from the mitochondria inan oocyte. A mean Δψ_(m) of approximately −119 millivolts is seen inhealthy, normal cells. See, e.g., Distelmaier et al., Ibid.

Oocytes with diminished Δψ_(m) are microinjected with phosphocreatine tosupplement cytoplasmic energy stores. Methods to microinject mammalianoocytes are know in the art (see e.g., U.S. Patent Application No.2003/0157086, which is incorporated herein by reference). Formicroinjection, six picoliters of 330 millimolar phosphocreatine(available from Sigma-Aldrich, St. Louis, Mo.) is microinjected intosingle oocytes using a Zeiss Axiovert 135 inverted microscope equippedwith Narishige micromanipulators and a PL-100 pico-injector (microscope,micromanipulators and injector are available from Tritech Research, LosAngeles, Calif.). Oocytes are cultured in media comprised of follicularfluid (obtained during follicle aspiration) and a balanced saltsolution, e.g., Ham's F10 media. Oocytes are cultured at about 37° C. inapproximately 5% CO₂ and 95% air for about 4-8 hours (see e.g., U.S.Pat. No. 5,563,059, which is incorporated herein by reference). Oocytequality is assessed by measuring Δψ, and by microscopic inspection (Seee.g., Van Blerkom et al., Human Rep. 10: 415-424, 1995 which isincorporated herein by reference). Normal appearing oocytes with anormal distribution of cytoplasmic organelles and a Δψ_(m) of about −119millivolts are selected for in vitro fertilization, andcryopreservation.

Oocyte quality is assessed by measuring Δψ_(m) and by microscopicinspection (See e.g., Van Blerkom et al., Human Rep. 10: 415-424 (1995),which is incorporated herein by reference). Normal appearing oocyteswith a normal distribution of cytoplasmic organelles, and a Δψ_(m) of−119 millivolts are selected for in vitro fertilization andcryopreservation.

Selected oocytes are fertilized in vitro with approximately 100,000spermatozoa per oocyte. Methods to collect and prepare spermatozoa andto inseminate oocytes are known in the art (see e.g., Rossato et al.,Ibid.). Fertilization is evaluated microscopically after 16-20 hours byidentification of pronuclei, and 46-48 hours after insemination theembryos are classified based on the number and shape of blastomeres, aswell as the percentage of fragments. Alternatively, healthy oocytes witha Δψ_(m) of approximately −119 millivolts are frozen for future use.Germinal vesicle, metaphase I oocytes, and metaphase II oocytes arefrozen using a slow freezing method (see e.g., Tao and Del Valle, J.Assist. Reprod. Genet. 25: 287-296, 2008, which is incorporated hereinby reference). Oocytes are treated with a cryoprotectant (e.g., dimethylsulfoxide, available from Sigma-Aldrich, St. Louis, Mo.), and thencooled to −7° C. at a rate of −1° C./min; then cooled to −35° C. at−0.3° C./min; then cooled rapidly to −80° C. and ultimately plunged intoliquid nitrogen (at −196° C.). Each oocyte is stored under liquidnitrogen in a microculture dish with its individual well locationreferenced to a computer address. Information stored on the computerincludes: the oocyte's microscopic evaluation, Δψ_(m), the femalesubject's identity, medical history, and genetic information.

Example 4 Implanted Devices to Supply Lipid Encapsulated ATP to Oocytesin vivo to Improve the Quality and Quantity of Oocytes Retrieved for invitro Fertilization or Cryopreservation

A female subject of advanced maternal age, 40 years of age, is treatedwith hormones to stimulate follicle development in her ovaries. Methodsto stimulate multiple ovarian follicles and isolate multiple oocytes areknown in the art (see e.g., Rossato et al., Hum. Reprod. 14: 694-697(1999), which is incorporated herein by reference). Follicle developmentis stimulated by administering a gonadotrophin releasing hormone analog(e.g., triptorelin, Trelstar La available from Watson Pharma, Inc.,Corona, Calif.), and follicle stimulating hormone (FSH; e.g., Follistim®AQ available from Schering Plough, Kenilworth, N.J.). FSH treatments aregiven for approximately 9 days and the follicles are monitored byvaginal ultrasound until antral follicles are apparent. Guided byvaginal ultrasound imaging, each antral follicle is injected with amicro-electromechanical system (MEMS) device using a dedicated syringe(available from Irvine Scientific, Santa Ana, Calif.). The MEMS devicecontains a sensor for an oocyte indicator, a reservoir, a nanopump todeliver liposomal ATP, and a RFID tag. The MEMS sensor detects NADHlevels in follicular fluid, an indicator of oocyte health whichcorrelates with levels of intracellular ATP (see e.g., Nadlinger et al.,Biochim. et Biophys. Acta 1573: 177-182 (2003), which is incorporatedherein by reference). Circuitry on the MEMS device detects diminishedlevels of follicular NADH, and signals delivery of liposomes containingATP to the oocyte. The oocyte indicator is monitored twice daily by theMEMS device and additional doses of liposomal ATP are delivered asrequired. The MEMS device contains a RFID tag that identifies thefollicle, and also links data on oxygen levels and liposomal ATPdelivered to a specific follicle. A RFID reader and remote computingdevice store and evaluate the cumulative data for each follicle and itsassociated oocyte.

Micro-electromechanical system devices are injected into ovarianfollicles to sense an indicator of oocyte health and to deliverliposomal ATP. A MEMS device is constructed with a NADH sensor to assessthe NADH concentration in follicular fluid. For example, an implantedmicrosensor that detects biological chemicals and transmits data to anexternal receiver is known in the art (see e.g., U.S. Pat. No.7,010,340, which is incorporated herein by reference). Moreover sensorsfor in vivo detection of NADH based on fluorimetry are described (seee.g., Mayevsky and Rogatsky, Am. J. Phys. 292: 615-640 (2007), which isincorporated herein by reference). A wireless, implantable MEMS device(see e.g., Product Sheet from Integrated Sensing Systems, Ypsilanti, MIwhich is incorporated herein by reference) is transvaginally injectedinto each follicle guided by ultrasound imaging, and the NADHconcentration in individual follicles is transmitted wirelessly to acomputing device. The NADH concentration for each follicle is measuredtwice daily, and the data are stored and analyzed on a computing device.Circuitry on the MEMS device detects electronic signals from the NADHsensor corresponding to changes in NADH concentration, and actuates ananopump to deliver liposomal ATP contained in a reservoir on the MEMSdevice. The nanopump delivers a liposomal ATP suspension at a flow rateof approximately 50 milliliters/(min.cm²) to the follicular fluid andthe oocyte (see e.g., U.S. Pat. No. 7,540,717, which is incorporatedherein by reference).

Liposomes containing 36-38 mole percent of ATP ((moles of ATP/moles oflipid)×100) are administered to deliver approximately 2 pmole of ATP tothe cytoplasm of the oocyte. Methods to prepare liposomes containing ATPare known in the art (see e.g., Liang et al., J. Microencap., 21:251-261 (2004); and U.S. Pat. No. 7,056,529, each of which areincorporated herein by reference). Liposomes comprised of phosphatidylcholine and cholesterol are combined with 400 mM ATP solution andsubjected to a freeze thaw process, and subsequently purified (see e.g.,Liang et al., Ibid.; lipids and ATP are available from Sigma-Aldrich,St. Louis, Mo.). Alternatively, liposomes loaded with ATP arecommercially available from FormuMax Scientific Inc., Palo Alto, Calif.

Follicular development is monitored using vaginal ultrasonography andoocytes are retrieved by aspiration of the follicles. When follicles areapproximately 18 mm in diameter human chorionic gonadotrophin (HCG,10,000 IU; e.g., Pregnyl®, available from Schering Plough, Kenilworth,N.J.) is administered, and approximately 36 hours after HCGadministration the oocytes are retrieved by transvaginal aspiration.Procedures and devices for transvaginal ultrasonography and aspirationof ovarian follicles are known in the art (see e.g., Wang and Sauer,Therap. and Clin. Risk Mgt, 2: 355-364(2006), which is incorporatedherein by reference). Follicular fluid containing an oocyte and the MEMSdevice with a micro RFID tag (see e.g., Product Sheet from HitachiEurope, Ltd., Maidenhead, Berkshire, U.K., which is incorporated hereinby reference) is cultured in vitro prior to in vitro fertilization ofthe oocyte. A RFID reader (available from Hitachi Europe, Ltd.,Maidenhead, Berkshire, U.K.) is used to detect the MEMS device, as wellas link the follicle and the corresponding oocyte with the accumulateddata on NADH levels and the amount of liposomal ATP delivered. Moreoverthe RFID tag identifies the female subject who produced the oocyte aswell as the associated data on her medical history, genetics, and thedose and schedule of her fertility drugs.

Individual oocytes are tested for their oxygen respiration rate using areal-time, noninvasive system (see e.g., Scott et al., Rep. Biomed.Online 17: 461-469 (2008), which is incorporated herein by reference).Oocytes are classified microscopically as immature (germinal vesicle andmetaphase I oocytes), or metaphase II oocytes. Oocytes are washed freeof all cannulus and granulosa cells, and placed in individual wells of aglass tray in Sage Complete Embryo Culture medium (available from SageIn vitro Fertilization, Inc., Trumbull, Conn.). Oxygen respiration ratemeasurements are made with microelectrodes that measure the steady-statediffusive oxygen flux toward oocytes placed in the bottom of narrowwells in culture trays. An instrument for measuring respiration inoocytes is commercially available, for example, EmbryoScope™, UnisenseFertilitech, Aarhus, Denmark. A baseline respiration rate (BRR) iscalculated from repeated measurement of the oxygen concentrationgradients in the well over an initial 5 hour time period. The mean BRRfor oocytes from female subjects greater than 40 years old issignificantly lower (P<0.01) than that for female subjects less than 35years of age (BRR=0.461 nL O₂/hr versus BRR=0.575 nL O₂/hr respectively;see e.g., Scott et al., Ibid.) In addition, all oocytes are tested todetermine their mitochondrial membrane potentials.

Mitochondrial membrane potential (Δψ_(m)) is a key indicator ofmitochondrial health and metabolic activity. Methods to measure Δψ_(m)in live cells are known in the art (see e.g., Distelmaier et al.,Cytometry Part A, 73A: 129-138 (2008), which is incorporated herein byreference). To measure Δψ_(m), oocytes are incubated with a fluorescentcationic dye, tetramethyl rhodamine methyl ester (TMRM; available fromLife Technologies Corp., Carlsbad, Calif.), that is taken up andsequestered by mitochondria. Oocytes are incubated in Medium 199(available from Life Technologies Corp., Carlsbad, Calif.) containing100 nM TMRM for about 25 minutes at 37° C., and then washed withphosphate buffered saline (PBS). To measure TMRM fluorescence, aninverted microscope (available from Carl Zeiss Microimaging, Inc.,Thornwood, N.Y.) with an X63 oil immersion objective is used, and theoocytes are maintained at 37° C. in a heated chamber. TMRM sequesteredwithin mitochondria is excited with 540 nm wavelength light forapproximately 100 milliseconds, and fluorescent images emitted atapproximately 565 nm are captured on a CoolSNAP HQ monochrome CCD-camera(available from Roper Scientific Photometrics, Vianen, The Netherlands).Image processing and analysis are performed using Image Pro Plus 5.1software (available from Media Cybernetics, Silver Spring, Md.) andOrigin Pro 7.5 software (available from Originlabs, Northampton, Mass.).Small changes in Δψ_(m) (e.g., approximately 12 millivolts) are seen asa reduction in average fluorescence intensity from the mitochondria inan oocyte. A mean Δψ_(m) of approximately −119 millivolts is seen forhealthy, normal cells. See, e.g., Distelmaier et al., Ibid.)

Oocytes are selected for fertilization and cryopreservation based ontheir NADH levels, respiration rate and Δψ_(m). Selected oocytes arefertilized in vitro with approximately 100,000 spermatozoa per oocyte.Methods to collect and prepare spermatozoa and to inseminate oocytes areknown in the art (see e.g., Rossato et al., Ibid.). Fertilization isevaluated microscopically after 16-20 hours by identification ofpronuclei, and 46-48 hours after insemination, the embryos areclassified for the number and shape of blastomeres and the percentage offragments. Alternatively, healthy oocytes with a Δψ_(m) of approximately−119 millivolts and a BRR equal to approximately 0.575 nL O₂/hr arefrozen for future use. Metaphase I oocytes and metaphase II oocytes arefrozen using a slow freezing method (see e.g., Tao and Del Valle, J.Assist. Reprod. Genet. 25: 287-296 (2008), which is incorporated hereinby reference). Oocytes are treated with a cryoprotectant (e.g.,dimethylsulfoxide available from Sigma-Aldrich, St. Louis, Mo.) and thencooled in a step-wise fashion, first to −7° C. at a rate of −1° C./min;then cooled to −35° C. at −0.3° C./min; then cooled rapidly to −80° C.,and ultimately plunged into liquid nitrogen (at −196° C.). Each oocyteis stored under liquid nitrogen in a microculture dish with its MEMSdevice, and RFID tag for identifying the oocyte and linking it toinformation stored on a computer. The information includes: the oocyte'smicroscopic evaluation, NADH levels, liposomal ATP delivered,respiration rate, Δψ_(m) and the female subject's identity, medicalhistory, and genetic information.

Example 5 A Method to Provide ATP to a Plant Embryo that Promotes Growthand Development of the Plant Embryo

A horticulturist injects maize (Zea mays) embryos with ATP to promotethe development of a high percentage of plant embryos that survive andgrow vigorously. Native plant embryos derive from pollination of ovulesand fusion of sperm and egg cells to form a zygote. The zygotic embryodevelops into a mature embryo or seed that stores food and energysupplies for use during germination and seedling growth.

In order to increase the level of the energy substrate, ATP infertilized maize embryos they are isolated, microinjected with ATP,cultured in vitro and planted. Fertilized embryos of maize are isolatedfrom Zea mays plants grown to maturity. Methods to isolate plant embryosare known in the art (see e.g., U.S. Pat. No. 6,300,543, which isincorporated herein by reference). Maize plants with silks 6-10 cm inlength are pollinated by hand and the plants are placed in a growthchamber for at least 16 hours to allow fertilization to occur. Ovariesare isolated by removing husks and silks from the cobs and cutting thecobs transversely in 3 cm segments. The segments are sterilized for 10minutes in 70% ethanol and rinsed in deionized water. Ovaries are thenremoved and mounted for sectioning.

Specimen blocks for use in the microtome are surface sterilized in 70%ethanol for 10 minutes, and the alcohol is evaporated in a laminar flowhood. A thin layer of adhesive, “Quik Set 404” (available from LocktiteCorp., Newington, Conn.), is used to immobilize the ovaries with theiradiaxial surface up. The blocks with attached ovaries are placed in aVibratome (available from Technical Products International., St. Louis,Mo.), and sectioned at a thickness of 200-400 μm. Microscopic inspectionof the sections is used to identify 250 μm to 300 μm slices containingembryo sacs. Sections containing embryo sacs are collected and placed ona modified Murashige-Skoggs medium with 0.4 mg/L L-asparagine, 0.1 mg/L6-benzylaminopurine (BAP) and 15% sucrose, pH 5.8. Media and cultureconditions for plant embryos are known in the art (see e.g., Hecht etal., Physiol. Plant 127: 803-816 (2001), which is incorporated herein byreference).

Zygotes contained in intact embryo sacs are microinjected with ATP topromote embryo development, viability and growth. For microinjection,one to six picoliters of 200 mM ATP(available from Sigma-Aldrich, St.Louis, Mo.) is microinjected into single oocytes using a Stemi SV11Stereomicroscope (available from Carl Zeiss Microimaging, Inc.,Thornwood, N.Y.) equipped with Narishige micromanipulators and a PL-100pico-injector (micromanipulators and injector are available from TritechResearch, Los Angeles, Calif.). Both the zygote and the central cell aremicroinjected with ATP and the embryo sacs are placed in culture forapproximately 5 days. Embryo sacs are cultured at 25° C. in the dark onmodified

Murashige-Skoggs medium with 0.4 mg/L L-asparagine, 0.1 mg/L6-benzylaminopurine (BAP) and 15% sucrose, pH 5.8. Microscopicinspection shows that without ATP microinjection approximately 44% ofthe embryo sacs give rise to embryos (see e.g., U.S. Pat. No. 6,300,543,Ibid.).

In order to grow plants from the microinjected zygotes, the embryo sacsare transferred to media lacking BAP, and cultured in vitro. Embryo sacsare cultured in Murashige-Skoggs medium with 0.4 mg/L L-asparagine and10% sucrose, pH 5.8 for 5 days at 25° C. in the dark, and thentransferred to the same medium containing only 3% sucrose for another 5days. When young shoots are approximately 1.5 cm long, they are exposedto light. After the embryos have grown into plantlets in vitro, they areplanted in sterilized soil or vermiculite and grown to maturity in agreenhouse. Microinjection of ATP into plant embryos may be used toimprove the viability and growth of crosses between distantly relatedplant species (see e.g., Raghavan, In Vitro Cell. Dev. Biol.-Plant 39:437-442 (2003), which is incorporated herein by reference); or toimprove the viability and growth of transgenic plants (see e.g., U.S.Pat. No. 6,300,543, Ibid.).

Example 6 A Method to Provide ATP to Mature Plant Embryos to PromoteEfficient Germination and Growth of Viable Seedlings

A horticulturist injects mature soybean embryos with ATP usingmicroneedles to improve the germination, viability and vigor of thesoybean seeds. Soybean embryos with seed coats are injected withmicroneedles that penetrate the seed coat and provide ATP to the embryoincluding the endosperm, cotyledons and hypocotyl. Microinjected soybeanseeds are sealed with a coating that prevents leakage, as well asprevents microbial contamination of the soybean embryos. Soybean seedswith optimal levels of ATP are planted in the field and tested forimproved vigor.

Soybean plants (Glycine max) are cultivated in a greenhouse, and intactseeds at various distinct developmental stages are harvested. Soybeanseeds at an early stage, middle stage and late stage of nutrient (e.g.,lipids and starch) storage corresponding to approximately 80 mg, 240 mgand 400 mg per seed, respectively, are placed in the wells of amicrotiter plate. Microneedles (see e.g., McAllister et al., Proc. Natl.Acad. Sci. USA 100: 13755-13760 (2003), which is incorporated herein byreference) approximately 30 μm in diameter and approximately 1000 μm inlength are inserted into the adaxial region of the plant embryos to apenetration depth of approximately 100 to 1000 μm past the seed coat.Methods for injecting soybean seeds are known in the art (see e.g.,Rolletschek et al., New Phytologist 167: 777-786 (2005), which isincorporated herein by reference). Soybean seeds with ATP levels greaterthan or equal to approximately 175 nmoles/g are optimal (see e.g.,Rolletschek et al., Ibid).

In order to achieve optimal ATP levels in the soybean embryoapproximately 3 μL of 14 mM ATP solution is injected into each seed.Soybean embryo ATP levels are determined by using high performanceliquid chromatography (HPLC) system with an Ultrasphere ODS 250×4.60 mmcolumn; a Gold 125 Solvent System and and a Gold 168 diode arraydetector (all are available from Beckman Instruments Inc., USA). Methodsto determine ATP in plant tissues are known in the art (see e.g., Liu etal., Food Technol. Biotechnol. 44: 531-534 (2006), which is incorporatedherein by reference).

Soybean seeds with optimal ATP levels are coated with a polymer solutionthat prevents ATP and other metabolites from leaking out of the seed andmicrobial pathogens from entering the seed. Methods for coating seedswith water insoluble polymers are known in the art (see e.g., U.S. Pat.No. 3,947,996, which is incorporated herein, by reference).

Soybean seeds with optimal levels of ATP are tested to assess theirgermination, viability and vigor. Methods to assess seed germination,viability and vigor are known in the art (see e.g., Alandadi et al., J.Food Agric. Envir. vol. 7: 420-426 (2009), which is incorporated hereinby reference). Soybean seeds that have been microinjected with ATP, asdescribed above, are compared with control soybean seeds that have beenmicroinjected with saline. Seeds from each group are tested in astandard laboratory germination test. Approximately 400 ATP injectedseeds and 400 control seeds are placed under moist conditions onblotters or rolled towels and are maintained at 25° C. for about 7 days.At the end of this period, the seedlings are counted and characterizedas normal or abnormal. The percentage germination is calculated from thenumber of normal seedlings, and the total number of seeds is evaluated.

Separate lots of seeds are also tested in a cold germination test: Theseeds are placed under moist conditions, rolled in towels, and chilledat 10° C. for 7 days and transferred to 25° C. for 4 days. Next, thenumber of normal seedlings is counted, and the percentage is calculatedof normal seedlings relative to the total number of seeds tested. Nativesoybean seeds without microinjection display cold germinationpercentages that range between approximately 75.25% and 85.00%.Moreover, ten normal seedlings from each lot of seed are selected atrandom and the lengths of the root and the primary stem are measured. Inorder to compare the “ATP-injected” and “saline-injected” soybean seeds,a seedling Vigor Index is calculated based on the mean lengths of theprimary stems (MPSL) and roots (MPRL) of the seedlings, and thepercentage germination(PG) [Vigor Index=(MPSL+MPRL)×PG]. Vigor Indexvalues for native soybean seeds (without microinjection) range between1105 and 1825 (see e.g., Alandadi et al., Ibid.)

In order to determine field emergence and related attributes, the seedsare sown on rows of 5 meters in length with inter- and intra-row spaceof 50 cm and 5 cm, respectively. In each plot, 600 seeds are sown with200 seeds in each row. Next, the number of seedlings is counted until 14days after sowing. The percentage of seeds that emerge by 14 days aftersowing is calculated. Native soybean seeds that have not been injecteddisplay field emergence percentages ranging between 31% and 41% (seee.g., Alandadi et al., Ibid.).

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1. A method of modifying a reproductive cell, comprising: activelyintroducing at least one exogenous energy supplying factor into at leastone intracellular compartment of a reproductive cell.
 2. The method ofclaim 1, wherein the actively introducing at least one exogenous energysupplying factor into at least one intracellular compartment of areproductive cell includes injecting or electroporating the reproductivecell.
 3. The method of claim 1, wherein the at least one exogenousenergy supplying factor includes at least one of exogenous creatine,exogenous phosphocreatine, exogenous creatine kinase, exogenouschlorophyll, exogenous lactate, exogenous glucose or other carbohydrate,exogenous calcium, exogenous ATP, exogenous ADP, exogenous AMP, or aprecursor thereof.
 4. The method of claim 1, wherein the intracellularcompartment of a reproductive cell includes at least one of a nucleolus,nucleus, ribosome, vesicle, rough endoplasmic reticulum, Golgiapparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria,vacuole, cytoplasm, lysosome, or centriole.
 5. The method of claim 1,wherein the reproductive cell is located in at least one of in situ, invitro, in vivo, in utero, in planta, in silico, or ex vivo.
 6. Themethod of claim 1, further comprising detecting at least one endogenousenergy supplying factor in the reproductive cell prior to delivering atleast one exogenous energy supplying factor thereto.
 7. The method ofclaim 6, wherein the at least one endogenous energy supplying factor isthe same factor as the exogenous energy supplying factor.
 8. The methodof claim 1, further comprising measuring at least one endogenous energysupplying factor in the reproductive cell subsequent to injecting atleast one exogenous energy supplying factor therein.
 9. The method ofclaim 1, wherein the at least one exogenous energy supplying factor isinjected in response to the presence or level of at least one endogenousenergy supplying factor in the reproductive cell.
 10. The method ofclaim 1, further comprising detecting or measuring the level of the atleast one exogenous energy supplying factor in the reproductive cell.11. The method of claim 1, wherein the at least one exogenous energysupplying factor is provided in response to the presence or level of atleast one exogenous energy supplying factor previously delivered to thereproductive cell.
 12. The method of claim 1, wherein the at least oneexogenous energy supplying factor is provided in response to thepresence or level of at least one reproductive cell indicator.
 13. Themethod of claim 12, wherein the at least one reproductive cell indicatorincludes at least one indicator of a property of the reproductive cell;a property of administering the at least one energy supplying factor tothe reproductive cell; a property of the modified reproductive cell; aproperty of administering at least one additional round of an energysupplying factor to a modified reproductive cell; reproductive cell ortissue apoptosis; reproductive cell division; reproductive cellcytoskeletal rearrangement; reproductive cell mitochondrial quality,quantity, or arrangement; reproductive cell fertilization success orfailure; or reproductive cell or tissue secretion.
 14. The method ofclaim 1, further comprising selecting at least one reproductive cell formodifying by injecting the at least one exogenous energy supplyingfactor.
 15. The method of claim 14, wherein the at least onereproductive cell is selected for modifying based on one or morereproductive cell indicators.
 16. The method of claim 15, wherein theproperty of the reproductive cell parameters includes at least one ofreproductive cell size, reproductive cell stage, reproductive cellquality, health of the subject from which the reproductive celloriginates, species of the subject from which the reproductive celloriginates, cleavage rate of the reproductive cell, metabolic profile ofthe reproductive cell, genomic profile of the reproductive cell,transcriptomic profile of the reproductive cell, or proteomic profile ofthe reproductive cell, or storage conditions of the reproductive cell.17. The method of claim 15, wherein the storage conditions of thereproductive cell include at least one of duration of storage time,storage temperature, storage size, reproductive cell dilution, orstorage solution(s).
 18. The method of claim 1, wherein the reproductivecell includes at least one of a germ cell, gametogonium, or gamete. 19.The method of claim 1, wherein the reproductive cell includes at leastone of an oogonium, primary oocyte, secondary oocyte, ootid, ovum, polarbody, follicular cell, cumulus cell, spermatogonia, primaryspermatocyte, secondary spermatocyte, spermatid, spermatozoa, Sertolicell, or Leydig cell.
 20. The method of claim 1, further comprisingselecting at least one reproductive cell for manipulation.
 21. Themethod of claim 20, wherein manipulation includes utilizing the selectedreproductive cell for at least one fertilization event.
 22. The methodof claim 20, wherein manipulation includes at least one of cell membranestripping, genetic modification, freezing, fusing with anotherreproductive cell, or fusing with a somatic cell.
 23. The method ofclaim 20, wherein selecting the at least one reproductive cell formanipulation is based at least in part on at least one of detectedexogenous energy supplying factor, or detected endogenous energysupplying factor.
 24. A method of restoring mitochondrial function in areproductive cell, comprising administering to a subject containing atleast one reproductive cell, an amount and for a time sufficient toincrease endogenous ATP levels of at least one of the subject'sreproductive cells, of at least one of an angiotensin receptor blockeror an angiotensin converting enzyme inhibitor.
 25. The method of claim24, wherein the angiotensin receptor inhibitor includes angiotensin-IIreceptor blocker (ARB). 26.-28. (canceled)
 29. A method of modifying anoocyte, comprising: introducing at least one exogenous energy supplyingfactor into at least one intracellular compartment of an oocyte.
 30. Themethod of claim 29, wherein introducing includes allowing at least oneexogenous energy supply factor to diffuse into the at least oneintracellular compartment of the oocyte.
 31. The method of claim 29,wherein introducing at least one exogenous energy supplying factorincludes liposomal introduction by way of endocytosis. 32.-57.(canceled)
 58. A modified reproductive cell, produced by the process ofactively introducing at least one exogenous energy supplying factor intoat least one intracellular compartment of a reproductive cell.
 59. Themodified reproductive cell of claim 58, wherein the at least oneexogenous energy supplying factor includes at least one of exogenouscreatine, exogenous phosphocreatine, exogenous creatine kinase,exogenous chlorophyll, exogenous lactate, exogenous glucose or othercarbohydrate, exogenous calcium or other mineral.
 60. The modifiedreproductive cell of claim 58, wherein the at least one exogenous energysupplying factor includes exogenous ATP, exogenous ADP, exogenous AMP,or a precursor thereof.
 61. The modified reproductive cell of claim 58,wherein the reproductive cell has not yet completed meiosis.
 62. Themodified reproductive cell of claim 58, wherein the reproductive cellhas not yet entered meiosis.
 63. The modified reproductive cell of claim58, wherein the reproductive cell has not yet completed mitosis.
 64. Themodified reproductive cell of claim 58, wherein the reproductive cellhas not yet entered mitosis.
 65. The modified reproductive cell of claim58, wherein the reproductive cell has not yet completed phase I ofmeiosis.
 66. The modified reproductive cell of claim 58, wherein thereproductive cell is located in at least one of in situ, in vitro, invivo, in utero, in planta, in silico, or ex vivo.
 67. The modifiedreproductive cell of claim 58, wherein the reproductive cell isimplantable or transplantable.
 68. The modified reproductive cell ofclaim 58, wherein the reproductive cell is implanted or transplantedinto at least one subject.
 69. The modified reproductive cell of claim58, wherein the reproductive cell is implanted or transplanted into atleast one subject subsequent to modification.
 70. The modifiedreproductive cell of claim 58, wherein the at least one subject includesat least one of a plant, alga, or animal.
 71. The modified reproductivecell of claim 58, wherein the at least one subject includes at least oneof a vertebrate or invertebrate.
 72. The modified reproductive cell ofclaim 58, wherein the at least one subject includes at least one of anamphibian, mammal, reptile, fish, or bird.
 73. The modified reproductivecell of claim 58, wherein the at least one subject includes at least onehuman.
 74. The modified reproductive cell of claim 58, wherein thereproductive cell includes at least one of a germ cell, gametogonium, orgamete.
 75. The modified reproductive cell of claim 58, wherein thereproductive cell includes at least one of an oogonium, primary oocyte,secondary oocyte, ootid, ovum, polar body, follicular cell, cumuluscell, spermatogonia, primary spermatocyte, secondary spermatocyte,spermatid, spermatozoa, Sertoli cell, or Leydig cell.
 76. The modifiedreproductive cell of claim 58, further comprising at least one detectionmaterial configured for detecting at least one exogenous energysupplying factor or at least one reproductive cell indicator.
 77. Themodified reproductive cell of claim 76, further comprising a scorecardfor evaluating the at least one reproductive cell based at least in parton the at least one detected exogenous energy supplying factor or the atleast one detected reproductive cell indicator.